US5571797A - Method of inducing gene expression by ionizing radiation - Google Patents
Method of inducing gene expression by ionizing radiation Download PDFInfo
- Publication number
- US5571797A US5571797A US08/241,863 US24186394A US5571797A US 5571797 A US5571797 A US 5571797A US 24186394 A US24186394 A US 24186394A US 5571797 A US5571797 A US 5571797A
- Authority
- US
- United States
- Prior art keywords
- cells
- jun
- promoter
- dna
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/525—Tumour necrosis factor [TNF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
Definitions
- the present invention relates to methods of regulating gene transcription and polypeptide expression by ionizing radiation.
- ionizing radiation has been postulated to induce multiple biological effects by direct interaction with DNA or through the formation of free radical species leading to DNA damage (Hall, 1988). These effects include gene mutations, malignant transformation, and cell killing. Although ionizing radiation has been demonstrated to induce expression of certain DNA repair genes in some prokaryotic and lower eukaryotic cells, little is known about the effects of ionizing radiation on the regulation of mammalian gene expression (Borek, 1985). Several studies have described changes in the pattern of protein synthesis observed after irradiation of mammalian cells.
- ionizing radiation treatment of human malignant melanoma cells is associated with induction of several unidentified proteins (Boothman, et al., 1989).
- Synthesis of cyclin and co-regulated polypeptides is suppressed by ionizing radiation in rat REF52 cells but not in oncogene-transformed REF52 cell lines (Lambert and Borek, 1988).
- Other studies have demonstrated that certain growth factors or cytokines may be involved in x-ray-induced DNA damage.
- platelet-derived growth factor is released from endothelial cells after irradiation (Witte, et al., 1989).
- Initiation of mRNA synthesis is a critical control point in the regulation of cellular processes and depends on binding of certain transcriptional regulatory factors to specific DNA sequences. However, little is known about the regulation of transcriptional control by ionizing radiation exposure in eukaryotic cells. The effects of ionizing radiation on posttranscriptional regulation of mammalian gene expression are also unknown.
- ionizing radiation is useful as a therapy. Methods to enhance the effects of radiation, thereby reducing the necessary dose, would greatly benefit cancer patients.
- ionizing radiation is specifically delivered to cells either by external sources, such as a Co-60 gamma source or X-irradiation through a linear accelerator, or through internal means, for example radioactively tagged monoclonal antibodies.
- a major problem with external irradiation is that many cancers are not localized to a limited area, but are spread throughout the body as distant metastases. Furthermore, monoclonal antibodies useful in internally irradiating cancerous tissue must be directed specifically to a particular tumor type.
- An important goal of the present invention was to develop alternative methods to specifically target ionizing radiation doses to tissues containing genes under the control of enhancers-promoters that are inducible by the radiation.
- the present invention seeks to overcome these and other drawbacks inherent in the prior art by providing methods of specifically irradiating tissues that contain radiation responsive enhancers-promoters operatively linked to structural genes encoding polypeptides having the ability to inhibit the growth of a cell, and in particular, a tumor cell.
- the invention overcomes the limitations by using the radioisotope Technetium (Tc99m), which may be chemically modified to preferentially accumulate in particular tissue types.
- Tc99m radioisotope Technetium
- Transfection of cells with radiation responsive enhancers-promoters operatively linked to a structural gene followed by treatment with Tc99m results in a two to twenty-fold increase in gene activation.
- the activation of promoter using this method is gradual and consistent compared to that obtained with X-rays, resulting in promoter activation over a longer duration, and in principle, at multiple metastatic sites.
- a radiation responsive enhancer-promoter comprises a CArG domain of an Egr-1 promoter, a TNF- ⁇ promoter or a c-Jun promoter.
- an encoding region encodes a single polypeptide.
- a preferred polypeptide encoded by such an encoding region has the ability to inhibit the growth of a cell and, particularly a tumor cell.
- An exemplary and preferred polypeptide is a cytokine, a dominant negative, a tumor suppressing factor, an angiogenesis inhibitor or a monocyte chemoattractant. More particularly, such a preferred polypeptide is TNF- ⁇ , interleukin-4, JE, ricin, PF4 Pseudomonas toxin, p53, the retinoblastoma gene product or the Wilms' tumor gene product.
- Another preferred polypeptide encoded by such an encoding region has radioprotective activity toward normal tissue.
- An exemplary and preferred such polypeptide having radioprotective activity is interleukin-1; TNF; a tissue growth factor such as a hematopoietic growth factor, a hepatocyte growth factor, a kidney growth factor, an endothelial growth factor or a vascular smooth muscle growth factor; interleukin-6; a free radical scavenger or a tissue growth factor receptor.
- Another preferred polypeptide encoded by such an encoding region has radioprotective activity toward normal tissue.
- An exemplary and preferred such polypeptide having radioprotective activity is interleukin-1; TNF; a tissue growth factor such as a hematopoietic growth factor, a hepatocyte growth factor, a kidney growth factor, an endothelial growth factor or a vascular smooth muscle growth factor; interleukin-6; a free radical scavenger or a tissue growth factor receptor.
- a hematopoietic growth factor is interleukin-3 or a colony stimulating factor (CSF) such as GM-CSF, G-CSF and M-CSF; 2) an endothelial growth factor is basic fibroblast growth factor (bFGF); 3) a vascular smooth muscle growth factor is platelet derived growth factor (PDGF); and 4) a free radical scavenger is manganese superoxide dismutase (MnSOD).
- CSF colony stimulating factor
- bFGF basic fibroblast growth factor
- PDGF platelet derived growth factor
- MnSOD manganese superoxide dismutase
- Yet another preferred polypeptide encoded by such an encoding region has anticoagulant, thrombolytic or thrombotic activity as exemplified by plasminogen activator, a streptokinase or a plasminogen activator inhibitor.
- a further preferred polypeptide encoded by such an encoding region has the ability to catalyze the conversion of a pro-drug to a drug.
- exemplary and preferred such polypeptides are herpes simplex virus thymidine kinase and a cytosine deaminase.
- a further preferred polypeptide encoded by such an encoding region is a surface antigen that is a gene product of a major histocompatibility complex.
- exemplary and preferred such polypeptides are H2 proteins and HLA protein.
- an encoding region of a DNA molecule of the present invention encodes the whole or a portion of more than one polypeptide.
- those polypeptides are transcription factors.
- an encoding region comprises:
- a first encoding sequence encodes a DNA binding domain of transcription factor GAL4
- a second encoding sequence encodes the VP-16 activation domain, the NF- ⁇ B activation domain, the repression domain of the Wilms' tumor suppressor gene WT1 or the repression domain of Egr-1.
- a DNA molecule of the present invention comprises a binding region that is capable of binding a DNA binding domain of a transcription factor, which binding region is operatively linked to a minimal promoter that is operatively linked to an encoding region that encodes a polypeptide, which encoding region is operatively linked to a transcription-terminating region.
- the transcription factor is GAL4 and the polypeptide is the same as set forth above.
- the present invention also contemplates a pharmaceutical composition
- a pharmaceutical composition comprising a DNA molecule of the present invention and a physiologically acceptable carrier.
- the present invention contemplates a cell transformed or transfected with a DNA molecule of this invention or a transgenic cell derived from such a transformed or transfected cell.
- a transformed or transgenic cell of the present invention is a leukocyte such as a tumor infiltrating lymphocyte or a T cell or a tumor cell.
- the present invention contemplates a process of regulating the expression of a polypeptide comprising the steps of:
- DNA molecules comprise:
- a first DNA molecule comprising a radiation responsive enhancer-promoter operatively linked to an encoding region that comprises:
- a second DNA molecule comprising a binding region that is capable of binding the DNA binding domain of the first transcription factor, which binding region is operatively linked to a minimal promoter that is operatively linked to an encoding region that encodes a polypeptide, which encoding region is operatively linked to a transcription-terminating region.
- a radiation responsive enhancer-promoter, a transcription factor, a binding domain of a transcription factor and an activation or repression domain of a transcription factor are preferably those set forth above.
- a polypeptide encoded by an encoding region is also preferably the same as set forth above.
- an encoding region preferably comprises:
- the second encoding sequence encodes the repression domain of the Wilms' tumor suppressor gene WT1 or the repression domain of Egr-1.
- the present invention contemplates a process of inhibiting growth of a tumor comprising the steps of:
- a radiation responsive enhancer-promoter comprises a CArG domain of an Egr-1 promoter, a TNF- ⁇ promoter or a c-Jun promoter and a polypeptide is a cytokine, a dominant negative, a tumor suppressing factor or an angiogenesis inhibitor.
- Delivering is preferably introducing the DNA molecule into the tumor. Where the tumor is in a subject, delivering is administering the DNA molecule into the circulatory system of the subject. In a preferred embodiment, administering comprises the steps of:
- a vehicle is preferably a cell transformed or transfected with the DNA molecule.
- An exemplary and preferred transformed or transfected cell is a leukocyte such as a tumor infiltrating lymphocyte or a T cell or a tumor cell from the tumor being treated.
- the vehicle is a virus or an antibody that immunoreacts with an antigen of the tumor.
- exposing comprises the steps of:
- a process of inhibiting growth of a tumor comprises the steps of:
- a first DNA molecule comprising a radiation responsive enhancer-promoter operatively linked to an encoding region that comprises:
- a second DNA molecule comprising a binding region that is capable of binding the DNA binding domain of the first transcription factor, which binding region is operatively linked to a minimal promoter that is operatively linked to an encoding region that encodes a polypeptide that has the ability to inhibit the growth of a tumor cell, which encoding region is operatively linked to a transcription-terminating region;
- a radiation responsive enhancer-promoter and a polypeptide are the same as set forth above.
- Delivering is preferably the same as set forth above.
- cells are exposed to ionizing radiation by delivery of radionuclides that specifically target, or that can be conjugated or otherwise modified to specifically target, tumor cells.
- Preferred radionuclides include, but are not limited to Technetium-99, Sodium Iodide-123, Sodium Iodide-125, Sodium Iodide-131, Potassium Chloride-42, Gold-198, or Sodium Phosphate-32, with Technetium-99 being specifically preferred.
- Intravenous dosages of radionuclides may range from 0.005 mCi to 100 mCi. In certain embodiments, the dose will preferably be in the range of 0.4 to 5 mCi.
- Radionuclides, or conjugates thereof are preferably supplied in a sterile, pyrogen-free solution suitable for intravenous administration. Alternatively, radionuclides may be administered orally or by topical application.
- Radionuclides suitable for the invention may be gamma or beta emitters.
- the radionuclide is a gamma emitter.
- Radionuclide Specific targeting of a radionuclide to a particular tissue or cell type may be accomplished through the use of derivatives of the radionuclide that have the effect of causing it to localize in specific tissues.
- derivatives of the radionuclide that have the effect of causing it to localize in specific tissues.
- preferred intravenous dosages of this radionuclide range from 1 to 4 mCi.
- Technetium-99m Sodium Methylene Diphosphonate is known to concentrate in areas of altered osteogenesis.
- preferred intravenous dosage is up to 4 mCi.
- the radionuclides may be administered directly to solid tumors.
- Cobalt-60 rods ensheathed in stainless steel may be embedded in the solid tumor that has been previously transfected with the radiation responsive enhancer-promoter.
- small, stainless steel ensheathed seeds of Iridium-192 embedded in a nylon ribbon may be employed as a therapeutic applicator of ionizing radiation.
- specific targeting to solid tumors will be accomplished by the administration by direct injection or infusion of radionuclides or a radionuclide conjugate composition to the site of the tumor.
- This method is particularly suitable for all solid tumors, the general location of which can be readily determined by a variety of means known to physicians.
- the ⁇ targeting ⁇ aspect of the invention comes from the generally specific administration of the radionuclides rather than any inherent physical or chemical properties thereof.
- Model systems to assess the killing of transformed (cancerous) cells are known to be predictive of success in human treatment regimens, partly as the cell types are essentially the same and all malignant cells simply proliferate, having little interaction with other systems. This is different to the problems found using other more interactive biological systems, such as, for example, when studying components of the immune system in isolation.
- FIG. 1 Egr-1 enhancer-promoter cloned into the XhoI and SacI restriction endonuclease sites of the luciferase reporter vector.
- FIG. 2 Stimulation of Erg-1-LUC by 131 I and Tc99m in human pancreatic cell line AsPC-1.
- FIG. 3 The effect of 14.3 MBq of Tc99m on stimulation of Erg-1-LUC in the human pancreatic cancer cell line AsPC-1.
- the present invention relates to compositions and methods for regulating transcription of an encoding DNA sequence and expression of a polypeptide encoded by that sequence.
- a composition of the present invention comprises one or more synthetic DNA molecules comprising an enhancer-promoter region that is responsive to ionizing radiation and an encoding region that encodes at least one polypeptide.
- control is exerted over transcription of an encoding DNA sequence by an enhancer-promoter region responsive to ionizing radiation.
- the enhancer-promoter region is used as a switch to selectively affect expression of a polypeptide encoded by that sequence.
- the regulation of specific polypeptide expression in a distinct target cell or tissue provides opportunities for therapeutic destruction, alteration, or inactivation of that cell or tissue.
- a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes.
- promoter includes what is referred to in the art as an upstream promoter region, a promoter region or a promoter of a generalized eukaryotic RNA Polymerase II transcription unit. Exemplary and preferred promoters are the TATA box, the CAAT box and GC-rich sequence elements.
- An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene).
- a major function of an enhancer is to increase the level of transcription of an encoding region in a cell that contains one or more transcription factors that bind to that enhancer.
- an enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
- the phrase "enhancer-promoter” means a composite unit that contains both enhancer and promoter elements.
- a "radiation responsive enhancer-promoter” indicates an enhancer-promoter whose transcription controlling function is affected by ionizing radiation.
- a radiation responsive enhancer-promoter of the present invention stimulates or increases the rate of transcription of an encoding region controlled by that enhancer-promoter.
- An exemplary and preferred enhancer-promoter for use in a DNA molecule of the present invention is a CArG domain of an Egr-1 promoter, a promoter for tumor necrosis factor-alpha (TNF- ⁇ ) gene or a c-Jun promoter.
- Egr-1 gene also known as zif/268, TIS-8, NFGI-A and Krox-24; Sukhatme, et al. 1988; Christy, et al., 1988; Milbrandt, 1987; Lemaire, et al., 1988; Lim, et al., 1987; Gessler, 1990
- Egr-1 gene encodes a 533-amino acid residue nuclear phosphoprotein with a Cys 2 -His 2 zinc finger domain that is partially homologous to the corresponding domain in the Wilms' tumor-susceptibility gene (Gessler, 1990).
- the Egr-1 protein binds to the DNA sequence CGCCCCCGC in a zinc-dependent manner and functions as a regulator of gene transcription (Christy, et al., 1989; Cao, et al., 1990; Lau, et al., 1987). Both mitogenic and differentiation signals have been shown to induce the rapid and transient expression of Egr-1 in a variety of cell types. Exposure of human HL-525 cells to x-rays was associated with increases in Egr-1 mRNA levels. Those increases were maximal at 3 hours and transient. Nuclear run-on assays demonstrated that this effect was related at least in part to activation of Egr-1 gene transcription.
- Sequences responsive to ionizing radiation-induced signals were determined by deletion analysis of the Egr-1 promoter. X-ray inducibility of the Egr-1 gene was conferred by a region containing six serum response or CC(A/T) 6 GG (CArG) domains or domains.
- a region encompassing the three distal or upstream CArG elements was functional in the x-ray response as sequential deletion of those three CArG domains progressively decreased the response.
- a single CArG domain was found to be sufficient to confer X-ray inducibility. Those results indicate that ionizing radiation induces Egr-1 transcription through one or more CArG domains.
- the Egr-1 promoter region extending from position -957 upstream to the transcription start site to position +248 was ligated to a chloramphenicol acetyl transferase (CAT) reporter gene to form plasmid pEgr-1 P1.2.
- the Egr-1 promoter region contains several putative cis elements including six CArG domains (Christy, et al., 1989; Qureshi, et al., 1991).
- Treatment of pEgr-1 P1.2 transfected cells with ionizing radiation was associated with a 4.1-fold increase in CAT activity as compared to transfected but unirradiated cells.
- x-ray inducibility of Egr-1 is likely mediated by sequences present between -550 and -50 of the Egr-1 promoter.
- x-ray inducibility of the Egr-1 gene is conferred by a region of the Egr-1 promoter that contains CArG domains.
- the six CArG domains of the Egr-1 promoter are located within a region of the Egr-1 promoter located about 960 nucleotide bases upstream from the transcription initiation site of the Egr-1 gene (reference).
- a single CArG domain is sufficient to confer radiation inducibility.
- a radiation responsive enhancer-promoter comprises at least one of the three most distal (i.e. upstream) CArG domains.
- CArG domain or serum response element is functional in inducing transcription of this gene in response to serum and other signals (Triesman, 1990).
- the CArG element is required for c-fos induction by both PKC-mediated signaling pathways and by growth factor-induced signals independent of PKC (Fisch, et al., 1987; Gilman, 1988; Buscher, et al., 1988; Sheng, et al., 1988; Stumpo, et al., 1988; Graham, et al., 1991).
- ionizing radiation may result in the modification of other proteins that interact with the SRF or CArG domain.
- SAP-1 and p62 TcF ternary complex factor
- SRF-DNA complexes Dalton, et al., 1992; Shaw, et al., 1989
- p62 DBF direct binding factor
- SRE-ZBP undergoes posttranslational modification and binds to this element (Attar, et al., 1992).
- One or more of these proteins may therefore be involved in x-ray-induced Egr-1 transcription.
- Exposure of cells to x-rays is associated with activation of the c-Jun/c-fos gene families, which encode transcription factors (Hallahan, et al., 1991; Sherman, et al., 1990).
- the c-Jun gene encodes the major form of the 40-44 kD AP-1 transcription factor (Mitchell, et al., 1989).
- the Jun/AP-1 complex binds to the heptomeric DNA consensus sequence TGA G / c TCA (Mitchell, et al., 1989).
- the DNA binding domain of c-Jun is shared by a family of transcription factors, including Jun-B, Jun-D and c-fos.
- the affinity of c-Jun binding to DNA is related to the formation of homodimers or heterodimers with products of the fos gene family (Zorial, et al., 1989; Nakabeppa, et al., 1988; Halazonetis, et al., 1988).
- Phorbol ester activation of c-Jun transcription in diverse cell types has implicated the involvement of a protein kinase C (PKC)-dependent mechanism (Brenner, et al., 1989; Angel, et al., 1988b; Hallahan, et al., 1991a).
- PKC protein kinase C
- a similar pathway likely plays a role, at least in part, in the induction of c-Jun expression by ionizing radiation.
- Prolonged treatment with phorbol esters to down-regulate PKC is associated with decreases in the effects of x-rays on c-Jun transcription (Hallahan, et al., 1991a).
- non-specific inhibitors of PKC such as the isoquinolinesulfonamide derivative, H7, block x-ray-induced c-Jun gene product expression (Hallaban, et al., 1991a).
- HL-525 which variant is deficient in PKC-mediated signal transduction
- That variant is resistant to both phorbol ester-induced differentiation and x-ray-induced TNF gene product expression (Hallahan, et al., 1991b; Homma, et al., 1986) and resistant to the induction of c-Jun gene product expression by phorbol esters.
- Jun likely results in increased transcription of the AP-1 binding site following ionizing radiation exposure.
- the plasmid p3 ⁇ TRE-CAT (containing three AP-1 sites upstream of the minimal tk promoter from plasmid pBLCAT2) was transfected into RIT-3 cells. Irradiation of p3 ⁇ TRE-CAT transfectants resulted in a 3-fold increase in CAT expression.
- CAT expression increased about 3-fold relative to transfected, non-irradiated cells.
- Transfection of those cells with a plasmid having a deletion of the AP-1 site located at +150-bp (-132/+170 ⁇ AP-1CAT) resulted in a loss of x-ray-mediated induction of CAT expression.
- activated AP-1 likely participates in the transcription of c-Jun and the AP-1 DNA sequence is likely sufficient and necessary to confer x-ray-mediated c-Jun gene induction.
- a detailed description of x-ray induced transcription of DNA molecules containing a c-jun promoter can be found hereinafter in Examples 2, 3, 5 and 6.
- Tumor necrosis factor ⁇ is a polypeptide mediator of the cellular immune response with pleiotropic activity. TNF- ⁇ acts directly on vascular endothelium to increase the adhesion of leukocytes during the inflammatory process (Bevelacqua, et al., 1989). This in vivo response to TNF- ⁇ was suggested to be responsible for hemorrhagic necrosis and regression of transplantable mouse and human tumors (Carswell, 1975). TNF- ⁇ also has a direct effect on human cancer cell lines in vitro, resulting in cell death and growth inhibition (Sugarman, et al., 1985; Old, 1985).
- TNF- ⁇ The cytotoxic effect of TNF- ⁇ correlates with free-radical formation, DNA fragmentation, and microtubule destruction (Matthews, et al., 1988; Rubin, et al., 1988; Scanlon, et al., 1989; Yamauchi, et al., 1989; Matthews, et al., 1987; Neale, et al., 1988).
- Cell lines that are resistant to oxidative damage by TNF- ⁇ also have elevated free-radical buffering capacity (Zimmerman, et al., 1989; Wong, et al., 1988).
- TNF- ⁇ causes hydroxyl radical production in cells sensitive to killing by TNF- ⁇ (Matthews, et al., 1987).
- Cell lines sensitive to the oxidative damage produced by TNF- ⁇ have diminished radical-buffering capacity after TNF- ⁇ is added (Yamauchi, et al., 1989).
- Lower levels of hydroxyl radicals have been measured in cells resistant to TNF- ⁇ cytotoxicity when compared with cells sensitive to TNF- ⁇ killing (Matthews, et al., 1987).
- TNF- ⁇ is increased after treatment with x-rays in certain human sarcoma cells (e.g., STSAR-13 and STSAR-48).
- TNF- ⁇ mRNA levels were substantially elevated 3 and 6 hours after irradiation of STSAR-13 and STSAR-48 cells.
- TNF- ⁇ mRNA levels in cell line STSAR-13 increased by >2.5-fold as measured by densitometry 3 hours after exposure to 500 cGy and then declined to baseline levels by 6 hours.
- TNF- ⁇ transcripts increased at 6 hours after irradiation in cell line STSAR-48, thus indicating some heterogeneity between cell lines in terms of the kinetics of TNF- ⁇ gene expression. In contrast, irradiation had no detectable effect on 7S RNA levels or expression of the polymerase ⁇ gene.
- TNF- ⁇ mRNA The increase in TNF- ⁇ mRNA was accompanied by an increased expression of TNF- ⁇ protein, which increase was accompanied by secretion of TNF- ⁇ protein into the medium in which those cells were grown.
- Levels of TNF- ⁇ in the medium of human tumor cell lines and fibroblasts were quantified before and after exposure to ionizing radiation.
- TNF- ⁇ Five of 13 human bone and soft tissue sarcoma cell lines (STSAR-5, -13, -33, -43, and -48) released TNF- ⁇ into the medium after irradiation, whereas TNF- ⁇ levels were not elevated in supernatant from normal human fibroblast cell lines (GM-1522 and NHF-235) and four human epithelial tumor cell lines (HN-SCC-68, SCC-61, SCC-25, and SQ-20B) after exposure to radiation.
- Tumor cell line STSAR-13 produced undetectable amounts of TNF- ⁇ before x-irradiation and 0.35 units/ml after x-ray exposure.
- TNF- ⁇ protein in the medium was first elevated at 20 hr after x-ray treatment, reached maximal levels at 3 days, and remained elevated beyond 5 days. Furthermore, supernatant from irradiated, but not control STSAR-33 cells, was cytotoxic to TNF- ⁇ -sensitive cell line SQ-20B.
- x-ray induced transcription of DNA molecules containing the TNF- ⁇ promoter can be found hereinafter in Example 1.
- a radiation responsive enhancer-promoter is operatively linked to an encoding region that encodes at least one polypeptide.
- operatively linked means that an enhancer-promoter is connected to an encoding region in such a way that the transcription of that encoding region is controlled and regulated by that enhancer-promoter.
- Means for operatively linking an enhancer-promoter to an encoding region are well known in the art. As is also well known in the art, the precise orientation and location relative to an encoding region whose transcription is controlled, is dependent inter alia upon the specific nature of the enhancer-promoter.
- a TATA box minimal promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a transcription initiation site.
- an enhancer can be located downstream from the initiation site and can be at a considerable distance from that site.
- an encoding region of a DNA molecule of the present invention encodes a single polypeptide.
- polypeptide means a polymer of amino acids connected by amide linkages, wherein the number of amino acid residues can range from about 5 to about one million.
- a polypeptide has from about 10 to about 1000 amino acid residues and, even more preferably from about 20 to about 500 amino residues.
- a polypeptide includes what is often referred to in the art as an oligopeptide (5-10 amino acid residues), a polypeptide (11-100 amino acid residues) and a protein (>100 amino acid residues).
- a polypeptide encoded by an encoding region can undergo post-translational modification to form conjugates with carbohydrates, lipids, nucleic acids and the like to form glycopolypeptides (e.g., glycoproteins), lipopolypeptides (e.g. lipoproteins) and other like conjugates.
- glycopolypeptides e.g., glycoproteins
- lipopolypeptides e.g. lipoproteins
- any polypeptide can be encoded by an encoding region of a DNA molecule of the present invention.
- An encoding region can comprise introns and exons so long as the encoding region comprises at least one open reading frame for transcription, translation and expression of that polypeptide.
- an encoding region can comprise a gene, a split gene or a cDNA molecule.
- the encoding region comprises a split gene (contains one or more introns)
- a cell transformed or transfected with a DNA molecule containing that split gene must have means for removing those introns and splicing together the exons in the RNA transcript from that DNA molecule if expression of that gene product is desired.
- a polypeptide encoded by an encoding region of a DNA molecule of the present invention interferes with the structural or functional integrity of a cell exposed to that polypeptide.
- a polypeptide has the ability to inhibit the growth of a cell and, particularly a tumor cell.
- a polypeptide is preferably a cytokine, a dominant negative, a tumor suppressing factor, an angiogenesis inhibitor, or a monocyte chemoattractant.
- Dominant negatives to cellular enzymes such as Raf-1 kinase are cytotoxic to human tumor cells (Qureshi, et al., 1991). Dominant negatives to oncogenes such as N-myc may also be effective in the treatment of cancer.
- tumor suppressor genes such as p53, the retinoblastoma (Rb) susceptibility gene, Wilms' tumor gene can be controlled by radiation.
- Transfection of p53 deficient tumor cells with a p53 expression vector abrogates cell growth (Johnson, et al., 1991).
- Tumor growth is angiogenesis-dependent and angiogenesis is directly or indirectly induced by the tumor.
- Induction of angiogenesis is an important step in carcinogenesis and in metastatic development.
- Angiogenesis is induced during the transition from hyperplasia to neoplasia. Since angiogenesis is necessary for tumor growth, any natural or synthetic antiangiogenic compound may have an antineoplastic potential.
- Inhibition of tumor angiogenesis through controlled expression of an anti-angiogenesis gene could play an important role in cancer treatment.
- Inhibitors of capillary endothelial cell proliferation and/or angiogenesis are a cartilage-derived inhibitor and platelet factor 4 (PF4) (reviewed in Neta, et al., 1991; Zucker, et al., 1991).
- PF4 platelet factor 4
- the mouse fibroblast gene is induced by PDGF.
- the fibroblast gene product, JE or monocyte chemoattractant protein-1 (MCP-1) is a member of a family of cytokine-like glycoproteins whose expression is induced by a mitogenic signal in monocytes, macrophages and T cells.
- JE has been identified, characterized and recombinantly produced from both mouse and human fibroblasts (Rollins et al., 1989).
- the mouse and human fibroblast gene products are designated mJE and hJE, respectively.
- MCP-1 or JE is a monocyte-specific chemoattractant in vitro that is structurally related to a family of proinflammatory cytokines such as macrophage inflammatory proteins.
- Exemplary and preferred polypeptides are tumor necrosis factor (TNF), interleukin-4, JE, PF4, ricin, a bacterial toxin such as Pseudomonas toxin; p53, the retinoblastoma gene product or the Wilms' tumor gene product.
- TNF tumor necrosis factor
- JE interleukin-4
- PF4 ricin
- a bacterial toxin such as Pseudomonas toxin
- p53 the retinoblastoma gene product or the Wilms' tumor gene product.
- a polypeptide encoded by an encoding region has radioprotective activity toward normal cells (i.e., the polypeptide protects a normal cell or tissue from a deleterious effect of radiation).
- exemplary and preferred polypeptides having radioprotective activity are interleukin-1; tumor necrosis factor; a tissue growth factor such as a hematopoietic growth factor, a hepatocyte growth factor, a kidney growth factor, an endothelial growth factor or a vascular smooth muscle growth factor; interleukin-6, a free radical scavenger or a tissue growth factor receptor.
- a hematopoietic growth factor is a colony stimulating factor such as GM-CSF, G-CSF, M-CSF or interleukin-3; 2) an endothelial growth factor is basic fibroblast growth factor; 3) a vascular smooth muscle growth factor is platelet derived growth factor (PDGF); and 4) a free radical scavenger is manganese superoxide dismutase (MnSOD).
- IL-1 induces several hematopoietic growth factors (GM-CSF, G-CSF, M-CSF, IL 3, and IL 6) which clearly contribute to the accelerated growth and differentiation of hematopoietic progenitor cells (Neta, et al, 1991).
- Uckun et al have examined the radioprotective effects of pre-total body irradiation (TBI) conditioning with recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF) in a large series of lethally irradiated mice (Uckun, et al, 1989).
- TBI pre-total body irradiation
- rG-CSF recombinant granulocyte colony-stimulating factor
- rGM-CSF recombinant granulocyte-macrophage CSF
- rG-CSF or rGM-CSF before TBI protects a significant fraction of mice from the lethal effects of LD 100/30 TBI (Waddick, et al., 1991).
- rG-CSF displayed a more potent radioprotective activity than rGM-CSF.
- the survival rate after lethal TBI was also significantly higher in mice receiving optimally radioprotective doses of rG-CSF as compared with mice receiving optimally radioprotective doses of rGM-CSF.
- Pretreatment with rG-CSF followed by rGM-CSF was slightly more effective than rG-CSF alone in supralethally irradiated mice but not in lethally irradiated mice. Neta et al.
- TNF may induce radioprotection through the production of manganese superoxide dismutase (MnSOD), which has been shown to be associated with radiation resistance in the T-cell line HUT-78 (Wong, et al., 1991).
- MnSOD manganese superoxide dismutase
- C-met is the receptor for hepatocyte growth factor and is activated during kidney and liver regeneration. These genes can be used to prevent radiation injury to these organs.
- Arteriovenous malformations in the cerebrum have been treated with radiosurgery.
- This technology involves the direction of high dose irradiation to the AVM.
- the intima of AVMs thickens through endothelial proliferation and the microvasculature is obliterated (Steiner, 1984).
- Endothelial and smooth muscle proliferation have been shown to be associated with the production of bFGF and PDGF.
- Clinical results may be improved by the addition of bFGF and PDGF.
- the polypeptide encoded by the encoding region has anticoagulant, thrombolytic or thrombotic activity as exemplified by plasminogen activator, a streptokinase or a plasminogen activator inhibitor.
- a polypeptide encoded by an encoding region has the ability to catalyze the conversion of a pro-drug to a drug or to sensitize a cell to a therapeutic agent.
- HSV herpes simplex virus
- tk thymidine kinase
- GCV antiviral agent ganciclovir
- cells manipulated to contain a gene for bacterial cytosine deaminase and to express that enzyme can catalyze the conversion of inactive, non-toxic 5'-fluorocytosine to the active cytotoxin 5-fluorouracil (Culver et al., 1992).
- a preferred polypeptide that has the ability to catalyze the conversion of a pro-drug to a drug or to sensitize a cell to a therapeutic agent is herpes simplex virus thymidine kinase or a cytosine deaminase.
- a further preferred polypeptide encoded by an encoding region is a surface antigen that is a gene product of a major histocompatibility complex (MHC).
- MHC represents a set of linked genetic loci involved in regulating the immune response. MHC gene products occur on cell surfaces where they act as antigenic markers for distinguishing self from non-self. Typically, MHC gene products are classified as being of a class I or Class II depending upon their function. MHCs from different animals have been given different and corresponding designations.
- human MHC gene products are designated by the prefix HL; mouse MHC gene products are designated by the prefix H-2; rat MHC gene products are designated by the prefix RT1 and chimpanzee MHC gene products are designated by the prefix ChLA.
- Exemplary and preferred human MHC gene products are class I antigens HLA-A, HLA-B and HLA-D and class II antigens HLA-Dr and HLA-Dc.
- an encoding region of a DNA molecule of the present invention encodes the whole or a portion of more than one polypeptide.
- those polypeptides are transcription factors.
- a transcription factor is a regulatory protein that binds to a specific DNA sequence (e.g., promoters and enhancers) and regulates transcription of an encoding DNA region.
- a transcription factor comprises a binding domain that binds to DNA (a DNA binding domain) and a regulatory domain that controls transcription. Where a regulatory domain activates transcription, that regulatory domain is designated an activation domain. Where that regulatory domain inhibits transcription, that regulatory domain is designated a repression domain.
- an encoding region comprises:
- operatively linked in frame means that encoding sequences are connected to one another such that an open reading frame is maintained between those sequences.
- Means for linking DNA encoding sequences in frame are well known in the art.
- DNA binding domains of transcription factors are well known in the art.
- Exemplary transcription factors known to contain a DNA binding domain are the GAL4, c-fos, c-Jun, lac1, trpR, CAP, TFIID, CTF, Spl, HSTF and NF- ⁇ B proteins.
- a DNA binding domain is derived from the GAL4 protein.
- the GAL4 protein is a transcription factor of yeast comprising 881 amino acid residues.
- the yeast protein GAL4 activates transcription of genes required for catabolism of galactose and melibiose.
- GAL4 comprises numerous discrete domains including a DNA binding domain (Marmorstein et al., 1992).
- the DNA sequences recognized by GAL4 are 17 base pairs (bp) in length, and each site binds a dimer of the protein. Four such sites, similar but not identical in sequence, are found in the upstream activating sequence (UAS G ) that mediates GAL4 activation of the GAL1 and GAL10 genes, for example (Marmorstein et al., 1992).
- the DNA-binding region of GAL4 has six cysteine residues, conserved among a set of homologous proteins, that coordinate two Zn 2+ ions in a bimetal-thiolate cluster.
- Residues 10-40 which form the metal binding domain, are a compact globular unit.
- Residues 1-9 and residues C-terminal to 41 are disordered (Marmorstein et al., 1992).
- the protein fragment binds to its DNA site as a symmetrical dimer.
- Each subunit folds into three distinct modules: a compact, metal-binding domain (residues 8-40), an extended linker (residues 41-49), and an ⁇ -helical dimerization element (residues 50-64).
- the metal-binding domain contacts three DNA base pairs in the major groove, and is therefore referred to as a recognition module (Marmorstein et al., 1992).
- the recognition module is held together by two metal ions, tetrahedrally coordinated by the six cysteines.
- the recognition module of GAL4 defines a class in the group of DNA-binding domains that have Zn 2+ as a structural element.
- Residues 50-64 form an amphipathic ⁇ -helix.
- the complete GAL4 molecule contains additional residues between 65 and 100 that contribute to dimer interactions and maintain the protein as a dimer even when it is not bound to DNA.
- the aminoacid sequence of GAL4 is consistent with a coiled-coil that may continue for one heptad repeat beyond the C terminus of GAL4 (1-65).
- residues 79-99 include three potentially ⁇ -helical heptad sequences.
- the intervening segment (residues 72-78) contains a proline.
- the full dimerization element of GAL4 could therefore share some structural features with the ⁇ helix-loop-helix ⁇ transcription factors.
- At least eleven other fungal DNA-binding proteins are known to contain repeated CX 2 CX 6 C sequences like those found in the GAL4 recognition module: LAC9, PPR1, QA-1F, QUTA1, ARGRII, HAP1, MAL63, LEU3, PUT3, and AMDR.
- the ⁇ loop ⁇ between the third and fourth cysteines is six residues long, but it is one residue shorter in MAL63, PDR1, PUT3 and AMDR, and several residues longer in LEU3.
- GAL4 residues 15-20, which are in closest proximity to DNA in the complex, are highly conserved in these homologues.
- Arg 15 and Lys 20, which form phosphate salt links that anchor the first helix of the recognition module to DNA are conserved in all but AMDR, which has His and Arg at these positions, respectively.
- Residue 19 is usually hydrophobic, and residue 17 is basic, except in QA-1F and LEU3.
- Lys 18, which makes base-specific contacts in the GAL4 complex, is conserved in all but three cases. In two of the exceptions (QA-1F and MAL63) it is Arg; in the other (PUT3), it is His.
- GAL4 and LAC9 bind to the same DNA sites; PPR1 recognizes sites with the CCG triplet separated by six, rather than 11, base pairs.
- GAL4 and LAC9 have similar amino-acid sequences in their linker and dimerization segments; the linker and dimerization elements of PPR1 bear no sequence similarity to those of GAL4, aside from the rough characteristics of their heptad regions (Marmorstein et al., 1992) .
- a first encoding sequence of a DNA molecule of the present invention encodes a DNA binding domain of GAL4.
- that binding domain comprises amino acid residue sequences 1 to about 147 of GAL4, which numerical designations refer to amino acid residue sequences numbered consecutively beginning at the amino terminus.
- a first encoding sequence comprises about 444 nucleotide base pairs of the GAL4 gene, which base pairs encode amino acid residue sequences 1 to 147 of GAL4.
- a first encoding sequence of a DNA molecule of the present invention encodes a DNA binding domain of GAL4 that comprises amino acid residue sequences 1 to about 65 of GAL4, which numerical designations refer to amino acid residue sequences numbered consecutively beginning at the amino terminus.
- a first encoding sequence comprises about 198 nucleotide base pairs of the GAL4 gene, which base pairs encode amino acid residues 1 to 65 of GAL4.
- Transcription factors having activation or repression domains are well known in the art.
- Exemplary transcription factors having activation domains are GAL4, c-Jun, viral protein VP-16, and nuclear factor NF- ⁇ B.
- GAL4 a protein of 881 amino acid residues, activates transcription of factors involved in carbohydrate metabolism of yeast.
- activation domains comprise (1) amino acid residues 94 to 106, (2) amino acid residues 148 to 196, and (3) amino acid residues 768 to 881, where amino acid residues are numbered consecutively beginning at the amino terminus (Marmorstein et al., 1992).
- a second encoding sequence encodes an activation domain of GAL4.
- Such an encoding sequence comprises nucleotide base sequences of about, 69, 147 and 342 base pairs, respectively that encode the activation domains set forth above.
- C-Jun is a major form of the 40 to 44 kD AP-1 transcription factor.
- a 1 an additional transcriptional activation domain is found near the N terminus adjacent to a region termed Delta ( ⁇ ) which is proposed to bind a cellular protein that inhibits the transcriptional activating properties of Jun (Baichwal, 1990 and Baichwal, 1991. Jun transcriptional activity can be conferred through either or both activation domains A 1 and A2.
- Plasmid pSG-Jun5-253 contained the SV40 promoter (not transcriptionally responsive to radiation) upstream of an encoding region that encoded a chimeric protein (GAL4-Jun) comprising a sequence for ⁇ , A 1 , and A 2 (Baichwal, 1990) and the DNA binding domain of the yeast GAL4 gene (the DNA binding domain of Jun was replaced with the DNA binding domain of the GAL4 gene, Baichwal, 1990).
- GAL4-Jun chimeric protein
- a second plasmid, G5BCAT was constructed to contain the DNA sequence that binds Gal4 protein placed 5' of the E1b TATA box and upstream of the CAT reporter gene (Baichwal, 1990).
- a DNA molecule of the present invention comprises a binding region that is capable of binding a DNA binding domain of a transcription factor, which binding region is operatively linked to a minimal promoter that is operatively linked to an encoding region that encodes a polypeptide, which encoding region is operatively linked to a transcription-terminating region.
- a binding region is capable of binding the DNA binding domain of the first transcription factor set forth above.
- the binding domain is a Gal4 binding domain
- a binding region of a DNA molecule binds that Gal4 binding domain.
- a binding region is operatively linked to a minimal promoter (e.g., a TATA box) that is operatively linked to an encoding region that encodes a polypeptide.
- a minimal promoter e.g., a TATA box
- An exemplary preferred DNA molecule comprising a binding region, minimal promoter and encoding region is plasmid pG5BCAT.
- an activation domain is an activation domain of viral protein VP-16 or nuclear factor NK-fB.
- Viral protein VP-16 is a 65 kD polypeptide of about 490 amino acid residues that is expressed during the immediate early phase of herpes simplex viral infection and activates transcription and subsequent expression of infected cell proteins (ICP) such as ICP4 (Trienzenberg et al., 1988).
- ICP infected cell proteins
- the activation domain of VP-16 comprises an amino acid residue sequence of about 78 amino acid residues located at the carboxy-terminus of VP16 (amino acid residues 413 to 490 as numbered from the amino-terminus).
- the activation domain of VP16 is further likely centered in a 61 amino acid residue sequence located from about residue 429 to about residue 456 (Trienzenberg et al., 1988).
- a second encoding sequence encodes amino acid residue sequences from about residue number 413 to about residue number 490 of VP16 and, more preferably from about residue number 429 to about residue number 456 of VP16.
- Nuclear factor NF- ⁇ B is a transcription factor.
- the activation domain of NF- ⁇ B comprises amino acid residue sequences from about residue position 414 to about residue position 515, numbered from the amino-terminus.
- a second encoding sequence preferably comprises nucleotide base pairs that encode amino acid residues from about residue position 414 to about residue position 515 of NF- ⁇ B (Ballard, 1992).
- At least one of the encoding sequences contains a nuclear localization signal.
- a nuclear localization signal permits the encoded transcription factor to enter the nucleus and interact with DNA in the nucleus.
- a nuclear localization signal is contained in the first or second encoding sequence.
- a nuclear localization signal is not present in a first or second encoding sequence such a signal is contained in a third encoding sequence.
- Nuclear localization signals are well known in the art.
- An exemplary and preferred such signal is derived from Simian Virus 40 (SV40) large T antigen.
- SV40 nuclear localization signal comprises an amino acid residue sequence of from about 7 to about 15 amino acid residues around a lysine (Lys) residue at position 128 of SV40 large T antigen (Kalderon et al. 1984).
- a nuclear localization signal comprises the amino acid residue sequence of SV40 extending from about residue position 126 to about residue position 132.
- RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
- mRNA messenger RNA
- a preferred transcription-terminating region used in a DNA molecule of the present invention comprises nucleotides 1533 to about 2157 of the human growth hormone (Seeburg, 1982).
- a DNA molecule of the present invention is prepared in accordance with standard techniques well known to a skilled worker in the art. First, DNA fragments containing the various regions of a desired DNA molecule are prepared or isolated. Those regions are then ligated to form a DNA molecule of this invention. Means for synthesizing, isolating and ligating DNA fragments are well known in the art.
- a desired DNA sequence is of about 200 or more nucleotides
- that sequence is typically obtained from tissues, cells or commercially available constructs (e.g. vectors or plasmids) known to contain that desired sequence.
- a cDNA clone of Egr-1 has been isolated and sequenced from mouse liver (Tsai-Morris, 1988).
- a cDNA of c-Jun has been isolated and sequenced from rat (Hatori, 1988 and Unlap, 1992).
- Sources known to contain encoding DNA regions that encode specific polypeptides are well known in the art. Table 1 below summarizes sources known to contain encoding DNA sequences for exemplary polypeptides.
- DNA sequence of interest is obtained from a cell or other organism
- total DNA is extracted from that organism or cell and fragmented using restriction enzymes.
- restriction enzymes The choice of what restriction enzyme or enzymes to use is dependent upon the desired DNA sequence being obtained.
- Particular DNA sequences of interest are then isolated, identified and purified using standard techniques well known in the art. If needed, an encoding DNA sequence can be amplified prior to isolation.
- a preferred means of amplifying a DNA sequence of interest is the polymerase chain reaction.
- Plasmid pE425-TNF comprises nucleotide bases from nucleotide position -425 to nucleotide position +65 (relative to the transcription start site) of the Egr-1 gene operatively linked to an encoding region that encodes TNF- ⁇ .
- pE425-TNF was constructed from plasmids pE-TNF, which contains TNF cDNA, and plasmid pE425-CAT, which contains the Egr-1 segment, a transcription-terminating region and a polyadenylation segment from CAT.
- pE-TNF was digested with the restriction enzyme Pst I to yield a 1.1 kilobase (kb) fragment containing TNF cDNA.
- pE425-CAT was digested with the restriction enzyme Hind III to yield a 3.3 kb fragment containing the CAT gene and a 3.2 kb segment containing the Egr-1 fragment and the polyadenylation signal from CAT.
- the 1.1 kb fragment from pE-TNF and the 3.2 kb fragment from pE425-CAT were blunt ended at the 3' overhang with T4 DNA polymerase and at the 5' overhang with Klenow using standard procedures well known in the art.
- pE425 and TNF cDNA were blunt-end ligated using T4 DNA ligase and T4 RNA ligase using standard procedures well known in the art to form pE425-TNF.
- Digestion of pE425-TNF with BamH1 and Hind II yielded 1.4 kb and 4.5 kb segments of the sense construct and a 0.9 kb segment of the antisense construct indicating the sense orientation of the plasmid.
- Plasmid pE425-CAT was prepared from an about 491 base pair fragment of the Egr-1 promoter, which fragment is located from nucleotide base -425 to nucleotide base +65 relative to the transcriptional start site and plasmid pCATm (Gius et al. 1990).
- the 491 base pair fragment of Egr-1 was obtained from plasmid p2.4, which contained a 2.4 kb fragment of the 5' flanking sequence of the Egr-1 gene (Tsai-Morris, 1988). Briefly, Balb/c 3T3 liver DNA was used to construct a ⁇ Fix genomic library using a well known partial fill-in cloning procedure. About 100,000 unamplified clones in E.
- coli strain JC7623 (rec B, rec C, sbc B; Winas et al., 1985) were screened with a 32 P-labeled Egr-1 plasmid OC 3.1 (Sukhatme et al., 1988) that contained a full length 3.1 kb cDNA insert.
- Membranes (GeneScreenPlus, New England Nuclear) were hybridized for about 16 hours at about 65° C. in 1 percent SDS, 10 percent dextran sulfate and 1M NaCl. The filters were washed to a final stringency of 65° C. in 0.2 ⁇ SSC.
- Autoradiographs were prepared by exposing the filters for about 18 hours at -70° C. with an intensifying screen. A single clone, designated mgEgr-1.1 was obtained, which clone hybridized to the extreme 5' 120 bp EcoRI-ApaI fragment from plasmid OC 3.1.
- a 2.4 kb PvuII-PvuII fragment and a 6.6 kb XbaI-XbaI fragment derived from mgEgr-1.1 were subcloned into the SmaI and XbaI sites of pUC13 and pUC18 (Promega Corp. Madison, Wis.), respectively, to form plasmids p2.4 and p6.6 respectively.
- nucleotide base position -957 to nucleotide base position +248 relative to the transcription start site was obtained from plasmid p2.4 to form plasmid pEgr-1 P1.2.
- a deletion mutant was constructed from pEgr-1 P1.2 using oligomers and polymerase chain reaction to form the 491 base pair fragment extending from nucleotide base position -425 to nucleotide base position +65.
- Plasmid pCAT3m was obtained from Dr. Laimonis A. Laimins, Howard Hughes Medical Institute Research Laboratories, University of Chicago, Chicago, Ill.). Plasmid pE-TNF was prepared in accordance with the procedure of Wong (Wong, 1985).
- Plasmid pE425-p53 comprises an about 491 base pair fragment of the Egr-1 promoter operatively linked to an encoding region for the tumor suppressing factor p53.
- pE425-p53 was constructed from a plasmid (pC53SN3; Diller, 1990) that contains p53 cDNA, and plasmid pE425-CAT, which contains the Egr-1 segment and a transcription-terminating region, the polyadenylation segment from CAT. Plasmid pE425-CAT was prepared as described above.
- Plasmid pE425-raf 301-1 comprises an about 491 base pair fragment of the Egr-1 promoter operatively linked to an encoding region for a serine/threonine-specific protein kinase product of an oncogene from a 3611 murine sarcoma cell.
- pE425-raf 301-1 was constructed from plasmids pMN301-1, which contains the raf dominant negative (Kolch, 1991), and pE425-CAT, which contains the Egr-1 segment and a transcription-terminating region, the polyadenylation segment from CAT.
- Plasmid pE425-MnSOD comprises an about 491 base pair fragment of the Egr-1 promoter operatively linked to an encoding region for the free-radical scavenger manganese superoxide dismutase (MnSOD).
- pE425-MnSOD was constructed from a plasmid nMnSOD #0664 (Genentech) (Wong, 1989) which contains MnSOD cDNA and pE425-CAT, which contains the Egr-1 segment and a transcription-terminating region, the polyadenylation segment from CAT.
- Plasmid G5-TNF comprises the DNA binding domain of the yeast GAL4 gene and the E1b minimal promoter TATA box operatively linked to an encoding region that encodes TNF- ⁇ .
- pG5-TNF was constructed from plasmid G5BCAT and plasmid pE-TNF.
- Plasmid G5BCAT which contains the DNA sequence which binds Gal4 protein placed 5' of the Elb TATA box upstream of the CAT reporter gene (Baichwal, 1990).
- the G5BCAT plasmid was digested with the EcoR1 restriction enzyme. The large fragment was isolated and blunt ended at the 3' overhang using T4 DNA polymerase and at the 5' overhang with Klenow. This digestion removes the minimal promoter but retains the poly-A end.
- TNF cDNA was removed from the pE4 plasmid using the Pst I restriction enzyme and the 1.1 kb fragment containing TNF cDNA was isolated and blunt ended at the 3' overhand using T4 DNA polymerase and at the 5' overhang with Klenow.
- the resulting G5B- and TNF cDNA were blunt-end ligated using the T4DNA ligase and T4 RNA ligase.
- the resulting G5-TNF plasmid underwent restriction enzyme mapping and DNA sequencing to assure the sense orientation of TNF.
- Plasmid G5BCAT was prepared by the method of Baichwal (Baichwal, et al., 1990). Plasmid pE-TNF was prepared as set forth above.
- Plasmid c-Jun-CAT comprises an about 1100 base pair fragment of the c-Jun promoter operatively linked to an encoding region for CAT. Plasmid c-Jun-CAT was constructed from plasmid h-jun-CAT in accordance with the procedure of Angel (Angel, 1988).
- a plasmid comprising a CArG domain of an Egr-1 promoter and an encoding region that encodes Pseudomonas exotoxin was prepared from plasmid PE425 and plasmid pMS150A (Lory, 1988), which contains the Pseudomonas exotoxin encoding region.
- DNA molecules of the present invention are made using techniques similar to those set forth above. Specific examples of the preparation of other DNA molecules can be found in Examples 1-6 hereinafter.
- the present invention contemplates a pharmaceutical composition comprising a therapeutically effective amount of at least one DNA molecule of the present invention and a physiologically acceptable carrier.
- a therapeutically effective amount of a DNA molecule that is combined with a carrier to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
- a specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
- a composition of the present invention is typically administered orally or parenterally in dosage unit formulations containing standard, well known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired.
- parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intraarterial injection, or infusion techniques.
- Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions are formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
- the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
- Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
- sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil can be employed including synthetic mono- or di-glycerides.
- fatty acids such as oleic acid find use in the preparation of injectables.
- a DNA molecule of the present invention can also be complexed with a poly(L-Lysine)(PLL)-protein conjugate such as a transferrin-PLL conjugate or an asialoorosomucoid-PLL conjugate.
- a poly(L-Lysine)(PLL)-protein conjugate such as a transferrin-PLL conjugate or an asialoorosomucoid-PLL conjugate.
- Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, syrups, solutions, suspensions, and elixirs containing inert diluents commonly used in the art, such as water.
- Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
- the present invention provides a cell transformed or transfected with one or more DNA molecules of the present invention as well as transgenic cells derived from those transformed or transfected cells.
- Means of transforming or transfecting cells with exogenous DNA molecules are well known in the art.
- a DNA molecule is introduced into a cell using standard transformation or transfection techniques well known in the art such as calcium-phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposomes and direct microinjection (Sambrook, Fritsch and Maniatis, 1989).
- transfection mediated by either calcium phosphate or DEAE-dextran The most widely used method is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transferred to the nucleus. Depending on the cell type, up to 20% of a population of cultured cells can be transfected at any one time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for experiments that require transient expression of the foreign DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that carry integrated copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays.
- protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacteria are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transferred to the nucleus.
- Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of the plasmid DNA randomly integrated into the host chromosome.
- Electroporation can be extremely efficient and can be used both for transient expression of clones genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
- Liposome transformation involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane.
- DNA that is coated with a synthetic cationic lipid can be introduced into cells by fusion.
- Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as low-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.
- the present invention contemplates a process of regulating the expression of a polypeptide.
- Polypeptide expression is regulated by stimulating or inhibiting transcription of an encoding region that encodes that polypeptide.
- a process of regulating polypeptide expression comprises the steps of:
- a DNA molecule used with such a method is a DNA molecule of the present invention as set forth above.
- the phrase "effective expression-inducing dose of ionizing radiation” means that dose of ionizing radiation needed to stimulate or turn on a radiation responsive enhancer-promoter of the present invention.
- the amount of ionizing radiation needed in a given cell depends inter alia upon the nature of that cell.
- an effective expression-inducing dose is less than a dose of ionizing radiation that causes cell damage or death directly.
- Means for determining an effective expression inducing amount are well known in the art.
- an effective expression inducing amount is from about 2 to about 20 Gray (Gy) administered at a rate of from about 0.5 to about 2 Gy/minute. Even more preferably, an effective expression inducing amount of ionizing radiation is from about 5 to about 15 Gy.
- ionizing radiation means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons).
- An exemplary and preferred ionizing radiation is an x-radiation.
- Means for delivering x-radiation to a target tissue or cell are well known in the art.
- Cells containing a DNA molecule of the present invention encoding a particular polypeptide express that polypeptide when exposed to ionizing radiation.
- Plasmid pEgr-1 P1.2 comprises a radiation responsive enhancer-promoter (the Egr-1 promoter region extending from position -957 upstream to the transcription start site to position +248) operatively linked to the CAT reporter gene.
- irradiation of pE425-CAT transfected cells was associated with a 3.6-fold induction of CAT activity compared to that in non-irradiated cells transfected with this construct.
- Egr-1 promoter constructs were next used to further define the x-ray responsive elements in pE425-CAT. Sequential deletion of the three distal CArGs progressively decreased CAT activity. Plasmid pE395-CAT (first CArG deleted) conferred x-ray inducibility to a lesser extent than pE425-CAT. Deletion of the first and second CArG domains (pE359-CAT) resulted in further decreases in CAT activity, while deletion of the first three CArG domains (pE342-CAT)) was associated with minimal increases in CAT activity.
- TNF- ⁇ protein expression was induced by ionizing radiation in cells transfected with plasmid pE425-TNF.
- SQ-20B, RIT-3 and HL-525 cells were transfected with plasmid pE425-TNF by DEAE precipitation.
- Transfected cells were exposed to 10 Gy of x-radiation at a rate of 1 Gy/minute.
- TNF- ⁇ expression was increased about 2-fold, 5-fold and 4-fold, respectively in SQ-20B, RIT-3 and HL-525 cells when compared to transfected, non-irradiated cells.
- CAT expression was induced by ionizing radiation in RIT-3 cells transfected with plasmid c-Jun-CAT, which plasmid comprises a 1100 base pair segment of the c-Jun promoter operatively linked to a CAT gene.
- Cells were co-transfected with an SV40 promoter- ⁇ galactosidase expression vector to control for transfection efficiency.
- Transfectants were irradiated (10 Gy, 1 Gy/min, GE Maxitron) 40 hours after transfection.
- CAT was extracted 6 hours after irradiation.
- CAT activity increased about 3-fold following irradiation of RIT-3 cells transfected with pc-Jun-CAT.
- ⁇ gal expression was not affected by radiation. Ionizing radiation did not increase CAT expression in cells transfected with a plasmid comprising the minimal Jun promoter (nucleotide base position-18 to nucleotide base position +170 relative to the transcription start site) operatively linked to CAT.
- ionizing radiation can be used as a trigger to regulate transcription of an encoding region in a DNA molecule of the present invention and expression of a polypeptide encoded by that region.
- polypeptide expression is regulated by the use of two DNA molecules.
- One of those DNA molecules comprises a radiation responsive enhancer-promoter operatively linked to an encoding region that comprises:
- a second DNA molecule comprises a binding region that is capable of binding the DNA binding domain of the first transcription factor, which binding region is operatively linked to a minimal promoter that is operatively linked to an encoding region that encodes a polypeptide, which encoding region is operatively linked to a transcription-terminating region.
- a radiation responsive enhancer-promoter, a transcription factor, a binding domain of a transcription factor and an activation or repressor domain of a transcription factor are preferably those set forth above.
- a polypeptide encoded by an encoding region is also preferably the same as set forth above.
- the first plasmid contained the SV40 promoter (not transcriptionally responsive to radiation) upstream of the coding sequence, for ⁇ , A1, and A2 regions of the activation domain of the Jun protein, wherein the DNA binding domain of Jun was replaced with the DNA binding domain of GAL4.
- a second plasmid, G5BCAT contained the DNA sequence which binds Gal4 protein linked to a minimal TK promoter upstream of the CAT reporter gene.
- Transfected cells were irradiated with 10 Gy of x-rays.
- CAT activity increased in the irradiated, transfected HeLa and RIT-3 cells as compared to transfected, non-irradiated cells.
- Plasmid pE425-Gal4/VP-16 comprises an about 491 base pair fragment of the Egr-1 promoter containing 6 CArG domains, which fragment is operatively linked to an encoding region comprising a first encoding sequence encoding DNA binding domain of Gal4 operatively linked in frame to a second encoding sequence encoding the activation domain of viral protein VP-16.
- Plasmid G5-TNF comprises a DNA segment that binds the Gal4 binding domain operatively linked to minimal promoter operatively linked to an encoding region that encodes TNF- ⁇ .
- RIT-3 cells were co-transfected with the pE425-Gal/VP16 and G5-TNF plasmids using lipofectin. Transfected cells were irradiated 36 hours following transfection and TNF was assayed 10 hours following irradiation. The concentration of intracellular TNF increased about 9-fold as compared to cells transfected with G5-TNF alone.
- an encoding region preferably comprises:
- the second encoding sequence encodes the repression domain of the Wilms' tumor suppressor gene WT1 or the repression domain of Egr-1.
- a radiation responsive enhancer-promoter and a first transcription factor are the same as set forth above.
- the present invention contemplates a process of inhibiting growth of a tumor comprising the steps of:
- a radiation responsive enhancer-promoter comprises a CArG domain of an Egr-1 promoter, a TNF- ⁇ promoter or a c-Jun promoter and a polypeptide having the ability to inhibit tumor cell growth is a cytokine, a dominant negative, a tumor suppressing factor or an angiogenesis inhibitor.
- exemplary and preferred polypeptides are TNF- ⁇ , interleukin-4, ricin, Pseudomonas toxin, p53, the retinoblastoma gene product or the Wilms' tumor gene product.
- TNF is cytotoxic to tumor cells.
- An interaction between TNF and radiation was found in 12 human epithelial tumor cell lines analyzed for cytotoxicity to TNF and synergistic killing by combining the two agents (Hallahan, et al., 1990; Hallahan, et al., 1989).
- TNF was found to have cytotoxic effects at concentrations of 10 to 1000 units/ml in ten of twelve tumor cell lines studied (Hallahan, et al., 1990; Hallahan, et al., 1989).
- synergistic or additive killing by TNF and x-rays was observed in seven of those ten cell lines.
- Ricin is a cytotoxin that inactivates mammalian ribosomes by catalyzing the cleavage of the N-glycosidic bond of 28S rRNA (Endo & Tsurngi, 1987). This enzyme is extremely toxic when given systemically, but may be localized to tumor through the use of radiation targeting of the gene encoding ricin.
- the transforming growth factor type alpha gene has been fused to modified Pseudomonas toxin gene from which the cell-recognition domain has been deleted (Chaudhary, et al., 1987).
- the chimeric gene has been expressed in Escherichia coli, and the chimeric protein, PE40-TGF-alpha, has been highly purified.
- PE40-TGF-alpha kills cells expressing epidermal growth factor receptors and has little activity against cells with few receptors. This chimeric protein might be useful in treating cancers that contain high numbers of epidermal growth factor receptors.
- the gene encoding pseudomonas toxin or its chimeric may be targeted by radiation to eliminate the potential systemic sequelae of this toxin.
- the nucleic acid segment may be DNA (double or single-stranded) or RNA (e.g., mRNA, tRNA, rRNA); it may also be a "coding segment", i.e., one that encodes a protein or polypeptide, or it may be an antisense nucleic acid molecule, such as antisense RNA that may function to disrupt gene expression.
- the nucleic acid segments may thus be genomic sequences, including exons or introns alone or exons and introns, or coding cDNA regions, or in fact any construct that one desires to transfer to a cancer cell or tissue.
- Suitable nucleic acid segments may also be in virtually any form, such as naked DNA or RNA, including linear nucleic acid molecules and plasmids, or as a functional insert within the genomes of various recombinant viruses, including viruses with DNA genomes and retroviruses.
- Delivering is preferably injecting the DNA molecule into the tumor.
- delivering is preferably administering the DNA molecule into the circulatory system of the subject.
- administering comprises the steps of:
- a vehicle is preferably a cell transformed or transfected with the DNA molecule or a transfected cell derived from such a transformed or transfected cell.
- An exemplary and preferred transformed or transfected cell is a leukocyte such as a tumor infiltrating lymphocyte or a T cell or a tumor cell from the tumor being treated. Means for transforming or transfecting a cell with a DNA molecule of the present invention are set forth above.
- nucleic acid transfer or delivery is often referred to as "gene therapy”.
- gene therapy initial efforts toward postnatal (somatic) gene therapy relied on indirect means of introducing genes into tissues, e.g., target cells were removed from the body, infected with viral vectors carrying recombinant genes, and implanted into the body. These type of techniques are generally referred to as ex vivo treatment protocols.
- Wolff et al. suggested several potential applications of the direct injection method, including (a) the treatment of heritable disorders of muscle, (b) the modification of non-muscle disorders through muscle tissue expression of therapeutic transgenes, (c) vaccine development, and (d) a reversible type of gene transfer, in which DNA is administered much like a conventional pharmaceutical treatment.
- Liu and coworkers recently showed that the direct injection method can be successfully applied to the problem of influenza vaccine development (Ulmer et al., 1993).
- Human lymphocytes can also be transfected with radiation-inducible plasmid constructs using existing technology including retroviral mediated gene transfer (Overell, et al., 1991; Fauser, 1991).
- LAK cells which tend to home in on the tumor site in question with some degree of preference though as is well known, they will also distribute themselves in the body in other locations, may be used to target tumors. Indeed, one of the most important advantages of the radiation inducible system is that only those LAK cells, which are in the radiation field will be activated and will have their exogenously introduced lymphokine genes activated. Thus, for the case of LAK cells, there is no particular need for any further targeting.
- the vehicle is a virus or an antibody that specifically infects or immunoreacts with an antigen of the tumor.
- Retroviruses used to deliver the constructs to the host target tissues generally are viruses in which the 3' LTR (linear transfer region) has been inactivated. That is, these are enhancer-less 3' LTR's, often referred to as SIN (self-inactivating viruses) because after productive infection into the host cell, the 3' LTR is transferred to the 5' end and both viral LTR's are inactive with respect to transcriptional activity.
- SIN self-inactivating viruses
- a use of these viruses well known to those skilled in the art is to clone genes for which the regulatory elements of the cloned gene are inserted in the space between the two LTR's.
- An advantage of a viral infection system is that it allows for a very high level of infection into the appropriate recipient cell, e.g., LAK cells.
- a radiation responsive enhancer-promoter which is 5' of the appropriate encoding region may be cloned into the virus using standard techniques well known in the art.
- viral vectors such as retroviral vectors, herpes simplex virus (U.S. Pat. No. 5,288,641, incorporated herein by reference), cytomegalovirus, and the like may be employed, as described by Miller (1992, incorporated herein by reference); as may recombinant adeno-associated virus (AAV vectors), such as those described by U.S. Pat. No. 5,139,941, incorporated herein by reference; and, particularly, recombinant adenoviral vectors.
- AAV vectors adeno-associated virus
- Techniques for preparing replication-defective infective viruses are well known in the art, as exemplified by Ghosh-Choudhury & Graham (1987); McGrory et al. (1988); and Gluzman et al. (1982), each incorporated herein by reference.
- the viral constructs are delivered into a host by any method that causes the constructs to reach the cells of the target tissue, while preserving the characteristics of the construct used in this invention.
- a rat glioma cell line, C6-BU-1 showed differential susceptibility to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), namely, all the HSV-1 strains tested so far persisted in this cell line but the HSV-2 strains did not (Sakihama, et al., 1991).
- C6-BU-1 cells consist of subpopulations heterogeneous in susceptibility to HSV-1 which may be possibly interchangeable.
- Retrovirus vectors should thus prove useful in the selective delivery of this killer gene to dividing tumor cells in the nervous system, where most endogenous cells are not dividing. Radiation will be used to enhance the specificity of delivery or activation of transcription of the tk gene only in irradiated areas.
- Antibodies have been used to target and deliver DNA molecules.
- An N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate readily forms a complex with plasmid DNA (Trubetskoy et al., 1992).
- a complex of monoclonal antibodies against a cell surface thrombomodulin conjugated with NPLL was used to target a foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line and mouse lung. Those targeted endothelial cells expressed the product encoded by that foreign DNA.
- a process of inhibiting growth of a tumor comprises the steps of:
- a first DNA molecule comprising a radiation responsive enhancer-promoter operatively linked to an encoding region that comprises
- a second DNA molecule comprising a binding region that is capable of binding the DNA binding domain of the first transcription factor, which binding region is operatively linked to a minimal promoter that is operatively linked to an encoding region that encodes a polypeptide having tumor cell cytotoxic activity, which encoding region is operatively linked to a transcription-terminating region;
- a radiation responsive enhancer-promoter comprises a CArG domain of an Egr-1 promoter or an AP-1 binding domain of a c-Jun promoter and the polypeptide having tumor cell cytotoxic activity is a cytokine, a dominant negative, a tumor suppressing factor, or an angiogenesis inhibitor as set forth above.
- Delivering is preferably the same as set forth above.
- TNF- ⁇ is increased after treatment with x-rays in certain human sarcoma cells.
- the increase in TNF- ⁇ mRNA is accompanied by the increased production of TNF- ⁇ protein.
- the induction of a cytotoxic protein by exposure of cells containing the TNF gene to x-rays was suspected when medium decanted from irradiated cultures of some human sarcoma cell lines was found to be cytotoxic to those cells as well as to other tumor cell lines.
- the level of TNF- ⁇ in the irradiated tumor cultures was elevated over that of nonirradiated cells when analyzed by the ELISA technique (Sariban, et al., 1988). Subsequent investigations showed that elevated TNF- ⁇ protein after irradiation potentiates x-ray killing of cells by an unusual previously undescribed mechanism (see Example 1).
- RNA from untreated cells (control) and irradiated cells was size-fractionated and hybridized to 32P-labeled TNF- ⁇ cDNA (STSAR-13) and PE4 plasmid containing TNF- ⁇ cDNA (STSAR-48).
- STSAR-13 32P-labeled TNF- ⁇ cDNA
- STSAR-48 PE4 plasmid containing TNF- ⁇ cDNA
- the tumor necrosis factor ⁇ is increased after treatment with x-rays. Both mRNA and TNF- ⁇ proteins were increased.
- the TNF- ⁇ gene is the first mammalian gene found to have increased expression after exposure to ionizing radiation. This gene is not categorized as a DNA repair gene.
- a DNA molecule of the present invention has uses other than inhibition of tumor growth. Exemplary such uses are summarized below in Table 3.
- TNF- ⁇ protein production after x-irradiation the levels of TNF- ⁇ in the medium of human tumor cell lines and fibroblasts were quantified by the ELISA technique (Sariban, et al., 1988) before and after exposure to 500-cGy x-rays.
- TNF- ⁇ levels were not elevated in supernatant from normal human fibroblast cell lines (GM-1522 and NHF-235) and four human epithelial tumor cell lines (HN-SCC-68, SCC-61, SCC-25, and SQ-20B) after exposure to radiation.
- the assay accurately measures TNF- ⁇ levels between 0.1 and 2.0 units per ml (2.3 ⁇ 10 6 units/mg) (Saribon, et al., 1988).
- Tumor cell line STSAR-13 produced undetectable amounts of TNF- ⁇ before x-irradiation and 0.35 units/ml after x-ray exposure.
- Cell lines STSAR-5 and -33 responded to x-irradiation with increases in TNF- ⁇ concentrations of >5- to 10-fold; however quantities above 2 units/ml exceeded the range of the assay (Saribon, et al., 1988).
- Cell lines STSAR-43 and -48 demonstrated increases in TNF- ⁇ of 1.5- to 3-fold (Table 4, below).
- TNF- ⁇ protein in the medium was first elevated at 20 hr after x-ray treatment, reached maximal levels at 3 days, and remained elevated beyond 5 days. Furthermore, supernatant from irradiated, but not control STSAR-33, was cytotoxic to TNF- ⁇ -sensitive cell line SQ-20B.
- TNF- ⁇ mRNA Increased levels of TNF- ⁇ mRNA were detected in the TNF- ⁇ -producing sarcoma cell lines after irradiation relative to unirradiated controls.
- TNF- ⁇ transcripts were present in unirradiated STSAR-13 and -48 cell lines.
- TNF- ⁇ mRNA levels in cell line STSAR-13 increased by >2.5-fold as measured by densitometry 3 hr after exposure to 500 cGy and then declined to baseline levels by 6 hours.
- These transcripts increased at 6 hours after irradiation in cell line STSAR-48, thus indicating some heterogeneity between cell lines in terms of the kinetics of TNF- ⁇ gene expression.
- irradiation had no detectable effect on 7S RNA levels or expression of the polymerase ⁇ gene.
- D0 The radiosensitivity
- TNF- ⁇ was added 20 hr before irradiation, the D 0 was 60.4 cGy.
- Surviving fractions were corrected for the reduced PE with TNF- ⁇ .
- the interaction between TNF- ⁇ and radiation in STSAR-33 cells was synergistic (Dewey, 1989).
- TNF- ⁇ and x-rays were irradiated 20 hr after TNF- ⁇ was added. These cell lines do not produce TNF- ⁇ in response to ionizing radiation.
- TNF- ⁇ 1000 units/ml
- SQ-20B SQ-20B
- SCC-61 SCC-61 cells
- the D 0 for cell line SQ-20B is 239 cGy.
- TNF- ⁇ 1000 units/ml
- the D 0 was 130.4 cGy.
- TNF- ⁇ a synergistic interaction (Dewey, 1979) between TNF- ⁇ and x-rays was demonstrated in this cell line.
- TNF- ⁇ added after irradiation did not enhance cell killing by radiation in cell lines SQ-20B.
- Nonlethal concentrations of TNF- ⁇ (10 units/ml) resulted in enhanced radiation killing in cell line HNSCC-68, providing evidence that TNF- ⁇ may sensitize some epithelial as well as mesenchymal tumor cell lines to radiation.
- Culture medium for epithelial tumor cells was 72.5% Dulbecco's modified Eagle's medium/22.5% Ham's nutrient mixture F-12 [DMEM/F-12 (3:1)]5% fetal bovine serum (FBS), transferrin at 5 ⁇ g/ml/10 -10 M cholera toxin/1.8 ⁇ 10 -4 M adenine, hydrocortisone at 0.4 ⁇ g/ml/2 ⁇ 10 -11 M triodo-L-thyronine/penicillin at 100 units/ml/streptomycin at 100 ⁇ g/ml.
- Culture medium for sarcoma cells was DMEM/F-12 (3:1)/20% FBS, penicillin at 100 units/ml/streptomycin at 100 ⁇ g/ml.
- TNF- ⁇ Protein Assay Human sarcoma cells were cultured as described above and grown to confluence. The medium was analyzed for TNF- ⁇ 3 days after feeding and again 1-3 days after irradiation. Thirteen established human sarcoma cell lines were irradiated with 500-centigray (cGy) x-rays with a 250-kV Maxitron generator (Weichselbaum, et al., 1988). TNF- ⁇ was measured by ELISA with two monoclonal antibodies that had distinct epitopes for TNF- ⁇ protein (Sariban, et al., 1988); the assay detects TNF- ⁇ from 0.1 to 2.0 units/ml.
- RNA Isolation and RNA Blot Analysis Total cellular RNA was isolated from cells by using the guanidine thiocyanate-lithium chloride method (Cathala, et al., 1983). RNA was size-fractionated by formaldehyde-1% agarose gel electrophoresis, transferred to nylon membranes (GeneScreenPlus, New England Nuclear), hybridized as previously described to the 1.7-kilobase (kb) BarnHi fragment of the PE4 plasmid containing TNF- ⁇ cDNA (19, 23), and autoradiographed for 16 days at -85° C. with intensifying screens.
- kb 1.7-kilobase
- RNA blot hybridization of TNF- ⁇ was analyzed after cellular irradiation with 500 cGy. Cells were washed with cold phosphate-buffered saline and placed in ice at each time interval. RNA was isolated at 3, 6, and 12 hr after irradiation.
- Another embodiment of a DNA molecule derives from the c-Jun protooncogene and related genes. Ionizing radiation regulates expression of the c-Jun protooncogene, and also of related genes c-fos and Jun-B.
- the protein product of c-Jun contains a DNA binding region that is shared by members of a family of transcription factors. Expression level after radiation is dose dependent.
- the c-Jun gene encodes a component of the AP-1 protein complex and is important in early signaling events involved in various cellular functions.
- the product of the protooncogene c-Jun recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to certain growth factors and phorbol esters (Bohmann, et al., 1987; Angel, et al., 1988).
- the product of the c-Jun protooncogene contains a highly conserved DNA binding domain shared by a family of mammalian transcription factors including Jun-B, Jun-D, c-fos, fos-B, fra-1 and the yeast GCN4 protein.
- c-Jun transcripts are degraded posttranscriptionally by a labile protein in irradiated cells. Posttranscriptional regulation of the gene's expression is described in Sherman, et al., 1990.
- RNA was isolated as described (29). RNA (20 ⁇ g per lane) was separated in an agarose/formaldehyde gel, transferred to a nitrocellulose filter, and hybridized to the following 32P-labeled DNA probes: (i) the 1.8-kilobase (kb) BamHI/EcoRI c-Jun cDNA (30); (ii) the 0.91-kb Sca I/Nco I c-fos DNA consisting of exons 3 and 4 (31); (iii) the 1.8-kb EcoRI Jun-B cDNA isolated from the p465.20 plasmid (32); and (iv) the 2.0-kb PstI ⁇ -actin cDNA purified from pA1 (33). The autoradiograms were scanned using an LKB UltroScan XL laser densitometer and analyzed using the LKB GelScan XL software package. The intensity of c-Jun hybridization was
- HL-60 cells were treated with ionizing radiation and nuclei were isolated after 3 hours.
- Newly elongated 32 P-labeled RNA transcripts were hybridized to plasmid DNAs containing various cloned inserts after digestion with restriction endonucleases as follows: (i) the 2.0-kb Pst I fragment of the chicken ⁇ -actin pA1 plasmid (positive control); (ii) the 1.1-kb BamHI insert of the human ⁇ -globin gene (negative control); and (iii) the 1.8-kb BamHI/EcoRI fragment of the human c-Jun cDNA from the pBluescript SK(+) plasmid.
- the digested DNA was run in a 1% agarose gel and transferred to nitrocellulose filters by the method of Southern. Hybridization was performed with 10 7 cpm of 32 P-labeled RNA per ml of hybridization buffer for 72 h at 42° C. Autoradiography was performed for 3 days and the autoradiograms were scanned as already described.
- Jun homodimers and Jun/fos heterodimers regulate transcription by binding to AP1 sites in certain promoter regions (Curran and Franza, 1988).
- the Jun and fos genes are induced following x-ray exposure in human myeloid leukemia cells suggests that nuclear signal transducers participate in the cellular response to ionizing radiation.
- Egr-1 and Jun genes are rapidly and transiently expressed in the absence of de novo protein synthesis after ionizing radiation exposure. Egr-1 and Jun are most likely involved in signal transduction following x-irradiation. Down-regulation of PKC by TPA and H7 is associated with attenuation of Egr-1 and Jun gene induction by ionizing radiation, implicating activation of PKC and subsequent induction of the Egr-1 and Jun genes as signaling events which initiate the mammalian cell phenotypic response to ionizing radiation injury.
- Control RNA from unirradiated cells demonstrated low but detectable levels of Egr-1 and Jun transcripts.
- Egr-1 expression increased in a dose dependent manner in irradiated cells. Levels were low but detectable after 3 Gy and increased in a dose dependent manner following 10 and 20 Gy. Twenty Gy was used in experiments examining the time course of gene expression so that transcripts were easily detectable. Cells remained viable as determined by trypan blue dye exclusion during this time course. A time dependent increase in Egr-1 and Jun mRNA levels was observed.
- SQ-20B cells demonstrated coordinate increases in Egr-1 and Jun expression by 30 minutes after irradiation that declined to baseline within 3 hours.
- Egr-1 transcript levels were increased over basal at 3 hours while Jun was increased at one hour and returned to basal at 3 hours in AG1522. Jun levels were increased at 6 hours in 293 cells while Egr-1 was increased at 3 hours and returned to basal levels by 6 hours.
- Egr-1 and Jun participated as immediate early genes after x-irradiation
- the effects of protein synthesis inhibition by cycloheximide were studied in cell lines 293 and SQ-20B after x-ray exposure. Cycloheximide treatment alone resulted in a low but detectable increase in Egr-1 and Jun transcripts normalized to 7S.
- the level of Egr-1 and Jun expression returned to baseline.
- SQ-20B cells pretreated with CHI demonstrated persistent elevation of Egr-1 at 3 hours and 293 cells demonstrated persistent elevation of Jun mRNA at 6 hours after irradiation thus indicating superinduction of these transcripts.
- mRNA levels of transcription factors Egr-1 and Jun increased following ionizing radiation exposure in a time and dose dependent manner.
- the potential importance of the induction of Egr-1 and Jun by ionizing radiation is illustrated by the recent finding that x-ray induction of the PDGF ⁇ chain stimulates proliferation of vascular endothelial cells (Witte, et al., 1989).
- PDGF has AP-1 and Egr-1 binding domains while TNF has elements similar to AP-1 and Egr-1 target sequences (Rorsman, et al., 1989; Economou, et al., 1989).
- X-ray induction of PDGF and TNF are likely regulated by Egr-1 and Jun.
- SQ-20B cells were preincubated with 100 ⁇ M H7 (1-(5-isoquinolinylsulfonyl)-2-methyl piperazine) or 100 ⁇ M HA1004 (N-[2-methyl-amino]ethyl)-5-isoquino-linesulfonamide) (Seikagaku America, Inc., St.
- the cellular response to ionizing radiation includes cell cycle-specific growth arrest, activation of DNA repair mechanisms and subsequent proliferation of surviving cells.
- the events responsible for the control of this response remain unclear.
- Recent studies have demonstrated that ionizing radiation exposure is associated with activation of certain immediate-early genes that code for transcription factors. These include members of the Jun/fos and early growth response (Egr) gene families (Sherman, et al., 1990; Hallahan, et al, 1991).
- Other studies have demonstrated that x-rays induce expression and DNA binding activity of the nuclear factor ⁇ B (NF- ⁇ B; Brach, et al., 1991).
- the activation of these transcription factors may represent transduction of early nuclear signals to longer term changes in gene expression which constitute the response to ionizing radiation.
- irradiation of diverse cell types is also associated with increased expression of the TNF, PDGF, FGF and interleukin-1 genes (Hallahan, et al., 1989; Witte, et al, 1989; Woloschak, et al., 1990; Sherman, et al., 1991).
- Expression of cytokines is conceivably involved in the repair and repopulation associated with x-ray-induced damage to tissues, and may explain some of the organismal effects of ionizing radiation (Hall, 1988).
- immediate-early transcription factors serve to induce these changes in gene expression.
- the present studies relate to mechanisms responsible for x-ray-induced activation of the Egr-1 gene (also known as zif/268, TIS-8, NFGI-A and Krox-24; Sukhatme, et al., 1988; Christy, et al., 1988; Milbrandt, 1987; Lemaire, et al., 1988; Lim, et al., 1987).
- the Egr-1 gene encodes a 533-amino acid nuclear phosphoprotein with a Cys 2 -His 2 zinc finger domain that is partially homologous to the corresponding domain in the Wilms tumor-susceptibility gene (Gessler, 1990).
- the Egr-1 protein binds to the DNA sequence CGCCCCCGC in a zinc-dependent manner and functions as a regulator of gene transcription (Christy, et al, 1989; Cao, et al., 1990; Gupta, et al, 1991). Both mitogenic and differentiation signals have been shown to induce the rapid and transient expression of Egr-1 in a variety of cell types.
- the Egr-1 gene is induced after mitogenic stimulation of Balb/c-3T3 cells by serum, PDGF or FGF (Lau, et al, 1987; Sukhatme, et al., 1987).
- the Egr-1 gene is also induced during: 1) cardiac and neuronal differentiation of the pluripotent EC line (Sukhatme, et al., 1988); and 2) monocytic differentiating of human myeloid leukemia cell lines (Kharbanda, et al., 1991; Bernstein, et al., 1991).
- Egr-1 transcripts A low but detectable level of 3.4-kb Egr-1 transcripts were present in untreated HL-525 cells. In contrast, treatment with ionizing radiation was associated with an increase in Egr-1 expression that was detectable at 1 hour. Maximal increases (18-fold) in Egr-1 mRNA levels were obtained at 3 hours, while longer intervals were associated with down-regulation to nearly that in control cells. This transient induction of Egr-1 expression occurred in the absence of significant changes in actin mRNA levels.
- the Egr-1 promoter region extending from position -957 upstream to the transcription start site to position +248 was ligated to the CAT reporter gene (plasmid pEgr-1 P1.2).
- This region contains several putative cis elements including two AP-1 sites and six CArG domains (Christy, et al., 1989; Gius, et al., 1990).
- Treatment of the pEgr-1P1.2 transfected cells with ionizing radiation was associated with a 4.1-fold increase in CAT activity as compared to transfected but unirradiated cells.
- Egr-1 promoter constructs were next used to further define the x-ray responsive elements in pE425. These constructs have been previously described and are shown schematically in Scheme 1. Sequential deletion of the three distal CArGs progressively decreased CAT activity. pE395 (first CArG deleted) conferred x-ray inducibility to a lesser extent than pE425. Deletion of the first and second (pE359) CArGs resulted in further decreases in CAT activity, while deletion of the first three CArG domains (pE342) was associated with little if any increases in CAT activity. Taken together, these findings supported the hypothesis that the three distal CArG elements confer x-ray inducibility of the Egr-1 gene.
- RNA was purified by the guanidine isothiocyanate-cesium chloride technique (Grosschedl, et al., 1985). The RNA was analyzed by electrophoresis through 1% agarose formaldehyde gels, transferred to nitrocellulose filters, and hybridized to the following 32 p-labeled DNA probes: 1) the 0.7-kb non-zinc finger insert of a murine Egr-1 cDNA (9); and 2) the 2.0-kb PstI insert of a chicken ⁇ -actin gene purified from the pA1 plasmid (Dignam, et al., 1983). Hybridizations were performed at 42° C.
- the digested DNAs were run in 1% agarose gels and transferred to nitrocellulose filters. Hybridizations were performed with 10 7 cpm of 32 P-labeled RNA/ml in 10 mM Tris-HCl, pH 7.5, 4 ⁇ SSC, 1 mM EDTA, 0.1% SDS, 2 ⁇ Denhardt's solution, 40% formamide, and 100 ⁇ g/ml yeast tRNA for 72 h at 42° C.
- the filters were washed in: a) 2 ⁇ SSC-0.1% SDS at 37° C. for 30 min; b) 200 ng/ml RNase A in 2 ⁇ SSC at room temperature for 5 min; and c) 0.1 ⁇ SSC-0.1% SDS at 42° C. for 30 min.
- pEgr-1P1.2, pE425, pE395, pE359, pE342, pE125, pE98 and pE70 constructs were prepared as described (26).
- pE425/250TK was constructed by cloning a HindIII-SmaI fragment from pE425, spanning the region -425 to -250 of the Egr-1 promoter, upstream of the herpes simplex virus thymidine kinase (HSV-TK) promoter in plasmid pTK35CAT (Homma, et al., 1986).
- pE395/250TK was constructed in the same manner using a HindIII-SmaI fragment from pE395.
- pSRE1TK contains the 5'-most distal or first CArG domain in the Egr-1 promoter along with seven base pairs of the 5' and 3' flanking sequences cloned into the SalI-BamHI site of pTK35CAT (Homma, et al., 1986).
- the constructs were transfected into cells using the DEAE-dextran technique (Treisman, et al., 1990).
- Tris-buffered saline solution 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na 2 HPO 4 , 0.7 mM CaCl 2 , and 0.7 mM MgCl 2 containing 0.4 mg DEAE-dextran and 8 ⁇ g plasmid, at 37° C. for 45 min.
- the cells were washed with media containing 10% FBS, resuspended in complete media and then incubated at 37° C.
- the organic layer containing the acetylated [ 14 C]chloramphenicol was separated by thin-layer chromatography using chloroform:methanol (95%:5%; v/v). Following autoradiography, both acetylated and unacetylated forms of [ 14 C]chloramphenicol were cut from the plates, and the conversion of chloramphenicol to the acetylated form was calculated by measurement of radioactivity in a ⁇ -scintillation counter.
- HL-525 cells responded to ionizing radiation with induction of Egr-1 mRNA levels. These findings indicated that ionizing radiation increases Egr-1 expression through signaling pathways distinct from those activated during induction of this gene in TPA-treated cells. Furthermore, the finding that x-ray-induced TNF gene expression is attenuated in the HL-525 line (Gilman, 1988) suggests that ionizing radiation induces the Egr-1 and TNF genes by distinct signaling pathways in these cells.
- Ionizing radiation produces a wide range of effects on cells which include induction of mutations, lethality, malignant transformation in some surviving cells, cell cycle arrest, and subsequent proliferation of cells.
- Jun a transcription factor that is central to tumor promotion, proliferation and cell cycle regulation, is activated by DNA damaging agents in mammalian cells (Devary, 1991 and Bernstein, 1989).
- One proposed mechanism of Jun activation is through dissociation of Jun from an inhibitor of Jun transcription (Baichwal, 1990).
- nuclear proteins were extracted from irradiated human sarcoma cell line RIT-3 cells at 5 minutes intervals for 30 minutes following exposure to 10 Gy.
- the AP-1, NF ⁇ B, SP-1 and CTF binding sequences labeled with 32 P were incubated with cell extracts. DNA-protein mixtures were then separated by electrophoresis.
- An increase in nuclear protein binding to AP-1 DNA sequences was found at 10 to 20 minutes following irradiation as compared to untreated control cells in electrophoretic mobility shift assay, whereas there was no increase in nuclear protein binding to NF- ⁇ B, SP-1, Oct-1 or CTF following irradiation of RIT-3 cells.
- DNA-protein complexing was not prevented by adding the inhibitor of protein synthesis cycloheximide, to cells prior to irradiation.
- AP-1 binding was eliminated when non-labeled AP-1 consensus sequence (Rauscher, 1988) competed for nuclear protein when added to extracts prior to the addition of labeled AP-1 and eliminated the banding produced by extracts from irradiated RIT-3 cells.
- a nonspecific DNA sequence (Oct-1) did not compete for nuclear protein binding.
- Jun and fos antisera Chos, 1991 and Stopera, 1992
- Jun and fos antisera were added to nuclear extracts prior to the addition of labeled AP-1 DNA sequences.
- the addition of antiserum to the DNA binding domains of fos (Ab-2) and Jun (CRB) resulted in a reduction in protein complexing to the AP-1 sequence following irradiation.
- Increasing concentrations of antiserum progressively reduced protein-DNA complexes accordingly.
- the plasmid (p3 ⁇ TRE-CAT) containing three AP-1 sites upstream of the minimal tk promoter (pBLCAT2) was transfected into RIT-3 cells. Irradiation of p3 ⁇ TRE-CAT transfectants resulted in a 3 fold increase in CAT expression.
- the 1840-base pair segment of the c-Jun promoter placed upstream of the chloramphenicol acetyl transferase (CAT) gene [Angel, 1988] was transfected into RIT-3 cells. Following transfection, cells were maintained in 0.2% fetal calf serum (FCS) and irradiated (10 Gy, 2 Gy/min) 40 hrs post-transfection and CAT was extracted 5 hours after irradiation.
- FCS chloramphenicol acetyl transferase
- Transfection of the -1.1 kb to +740-bp region of the c-Jun promoter demonstrated a 3-fold increase in gene expression following exposure to ionizing radiation.
- Transfection of the plasmid with a deletion of the AP-1 site located at +150-bp resulted in a loss of x-ray-mediated induction.
- Jun transcriptional activity can be conferred through either or both activation domains A 1 and A 2 .
- Phorbol ester treatment results in the modification of the Jun protein by a protein kinase C (PKC)-dependent phosphorylation of the A 1 region and thereby auto-induces transcription of c-Jun (Binetruy, 1991 and (Pulverer, 1991).
- PKC protein kinase C
- pSG-Jun5-253 contains the SV40 promoter, which is not transcriptionally responsive to radiation, upstream of the coding sequence for ⁇ , A 1 , and A 2 (Baichwal, 1990).
- the DNA binding domain of Jun was replaced with the DNA binding domain of the yeast GAL4 gene which encodes a protein involved in yeast transcriptional regulation (Baichwal, 1990).
- a second plasmid, G5BCAT contains the DNA sequence which binds Gal4 protein placed 5' of the Elb TATA box upstream of the CAT reporter gene (Baichwal, 1990). When the activation domain of Jun protein becomes transcriptionally active, the chimeric Gal-Jun protein, initiates CAT transcription following binding to the Gal4 binding sequence.
- the inhibitor of Jun transcription that binds to the ⁇ /A 1 domains represses the transcriptional activity of A 1 .
- the inhibitor of Jun transcription was originally defined in experiments where an excess of Jun competed for inhibitor and thereby allowed uninhibited Jun to increase transcription of the G5-CAT construct when compared to untreated control cells. Based on these results, HeLa cells are reported to contain the Jun inhibitor (Baichwal, 1990) whereas, in HepG2 cells do not. To determine whether the Jun inhibitor is present in RIT-3 cells, the expression vector CMV-Jun, which constitutively expresses c-Jun, was co-transfected with pSGJun5-235 and G5BCAT.
- Nuclear Extracts were prepared according to previously described methods (Schreiber, 1989) at 10,20,30, and 60 min. after irradiation.
- RIT-3 cells (10 6 ) were washed in 10 ml PBS, scraped, and pelleted by centrifugation at 1500 g for 5 min. The pellet was resuspended in 1 ml PBS, transferred into an Eppendorf tube and pelleted again for 15 sec. PBS was removed and the cell pellet resuspended in 400 ⁇ l of cold buffer A (10 mM HEPES pH 7.9; 10 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.5 mM PMSF).
- cold buffer A (10 mM HEPES pH 7.9; 10 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.5 mM PMSF).
- the cells were allowed to swell on ice for 15 min, followed by the addition of 25 ml of 10% NP-40. The mixture was centrifuged for 30 sec and the nuclear pellet resuspended in 50 ⁇ l ice-cold buffer B (20 mM HEPES pH 7.9; 0.4M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM PMSF) for 15 min and the nuclear extract centrifuged for 5 min. Protein content was determined by the Bradford method (Bio-Rad). The AP-1 consensus sequence DNA (BRL-GIBCO) was end-labeled with [ -32 P]dATP using DNA polymerase I Klenow fragment.
- Binding Assays were performed by incubating the end-labeled DNA (1 ng) with 10 ⁇ g nuclear protein, 75 mM KCl and 1 ⁇ g/ml poly (dI-dC) in a 20 ⁇ l reaction for 20 min at room temperature.
- Competition Assays were performed using oligonucleotides corresponding to known cis-acting elements AP-1, and Oct-1 (BRL-GIBCO) at a 100-fold molar excess as compared to the labeled fragments. The reaction products were separated by 5% polyacrylamide gel electrophoresis, dried and analyzed by autoradiography.
- A. Antiserum to human transcription factors c-Jun (amino acids 73-87, Ab-2, Oncogene Sci.), c-Jun, Jun-B, Jun-D (Cambridge Research, log OA-11-837), and c-fos (amino acids 4-17, Ab-2, Oncogene Sci and (amino acids 1-14, Lot OA-11-823, Cambridge Research) were added to 10 ⁇ g of nuclear extracts at a 1:200 dilution and incubated at 24° C. with rocking for one hour. DNA segments were added as described above followed by separation using electrophoretic mobility shift assays.
- Plasmids contain c-Jun-CAT, 3 ⁇ TRE-CAT, and ⁇ AP-1-CAT (2 ⁇ g) were co-transfected with a plasmid containing a CMV promoter linked to the ⁇ -galactosidase gene (1 ⁇ g) and 12 ⁇ g of carrier DNA into RIT-3 cells.
- Cells were transfected using Lipofectin Reagent (BRL-GIBCO) for 20 hrs., followed by the addition of medium with 0.2% fetal bovine serum. Transfectants were incubated for 40 h after transfection followed by treatment with 10 Gy (1 Gy/min, GE Maxitron) ionizing radiation and harvested by scraping 6 h later.
- CAT activity was determined by separating acetylated-[ 14 C]chloramphenicol by ascending chromatography. Scintillation counting of both the non-acetylated and acetylated forms of [ 14 C]chloramphenicol was used to quantify CAT activity. shown above are the results of: The 1.1 Kb segment of the c-Jun promoter linked to CAT (c-Jun-CAT). The 3 ⁇ TRE-CAT. The ⁇ AP-1-CAT plasmid in which the AP-1 sequence has been deleted from the c-Jun promoter. CAT activity is compared to nonirradiated and TPA treated transfectants. The mean and standard errors of 3 experiments are presented.
- Plasmids pSG-Jun5-253 (2 ⁇ g) and G5BCAT (4 ⁇ g) were co-transfected with a plasmid containing a CMV promoter linked to the ⁇ -galactosidase gene (1 ⁇ g) and 12 ⁇ g of carrier DNA into RIT-3 and HeLa cells using lipofectin. CAT activity is compared to nonirradiated and TPA treated transfectants. The mean and standard errors of 3 experiments are presented.
- Ionizing radiation has been postulated to induce activation of DNA repair mechanisms, cell cycle arrest in G 2 phase and lethality by either direct interaction with DNA or through the formation or reactive oxygen intermediates (ROI) which damage DNA.
- ROI reactive oxygen intermediates
- Recent studies have further suggested a role for the activation of immediate-early genes in the response to ionizing radiation. For example, exposure of cells to x-rays is associated with activation of the c-Jun/c-fos and Egr-1 gene families which code for transcription factors.
- Other studies have demonstrated that ionizing radiation induces expression and DNA binding activity of the nuclear factor ⁇ B (NF- ⁇ B).
- transcription factors likely represents a critical control point in transducing early nuclear signals to longer term changes in gene expression that reflect the response to x-ray-induced damage.
- cytokines including TNF, platelet-derived growth factor, fibroblast growth factor and interleukin-1.
- TNF tumor necrosis factor
- platelet-derived growth factor fibroblast growth factor
- interleukin-1 interleukin-1
- the c-Jun gene codes for the major form of the 40-44 kD AP-1 transcription factor. As observed in irradiated cells, this gene is induced as an immediate early event in response to phorbol esters and certain growth factors.
- the Jun/AP-1 complex binds to the heptomeric DNA consensus sequence TGA G / C TCA.
- the DNA binding domain of c-Jun is shared by a family of transcription factors, including Jun-B, Jun-D and c-fos.
- the affinity of c-Jun binding to DNA is related to the formation of homodimers or heterodimers with products of the fos gene family.
- Jun-B also forms dimers and binds to the AP-1 element, although the trans-acting properties of Jun-B differ from those of c-Jun. While the product of the Jun-D gene also interacts with c-fos and has similar binding properties to that of c-Jun, the function of Jun-D is unknown. Certain insights are available regarding the signals which contribute to the regulation of these genes. For example, the finding that phorbol esters activate c-Jun transcription in diverse cell types has implicated the involvement of a phosphorylation- dependent mechanism. A similar pathway appears to play a role, at least in part, in the induction of c-Jun expression by ionizing radiation.
- the present studies examined the effects of ionizing radiation on c-Jun expression in an HL-60 cell variant, designated HL-525, which is deficient in PKC-mediated signal transduction. This variant is resistant to both phorbol ester-induced differentiation and x-ray-induced TNF gene expression.
- the present results demonstrate that HL-525 cells are also resistant to the induction of c-Jun expression by phorbol esters.
- the results also demonstrate that treatment of these cells with ionizing radiation is associated with a superinduction of c-Jun mRNA levels compared to phorbol ester-responsive HL-60 cells.
- the findings indicate that this effect of ionizing radiation is related at least in part to the formation of reactive oxygen intermediates.
- Proteins encoded by members of the Jun gene family can form heterodimers with fos gene products. Consequently, the effects of x-rays on c-fos and fos-B expression were also studied.
- c-fos transcripts were present at low levels in HL-205 cells and there was little if any effect of ionizing radiation on expression of this gene. Similar findings were obtained for fos-B.
- x-ray exposure was associated with transient increases in transcripts for both of these genes. The kinetics of these increases in fos gene expression were similar to that obtained for members of the Jun gene family. Thus, activation of multiple Jun and fos genes could contribute to diverse nuclear signals in the response of cells to x-rays.
- HL-60 cells and other myeloid leukemia cells with TPA Treatment of HL-60 cells and other myeloid leukemia cells with TPA is associated with induction of the c-Jun gene. Similar effects were obtained in TPA-treated HL-205 cells. The response of these cells to TPA was associated with increases in c-Jun expression that were detectable at 6 hours and reached maximal levels by 24 hours. In contrast, similar exposures of HL-525 cells to TPA resulted in an increase in c-Jun expression which was transient at 12 hours and attenuated compared to that in the HL-205 link. These findings indicated that HL-525 cells are resistant at least in part to the effects of TPA on Jun/AP-1-mediated signaling events.
- NAC had no detectable effect on induction of the c-Jun gene in HL-525 cells by 1- ⁇ -D-arabinofuranosylcytosine (ara-C; data not shown), another DNA-damaging agent which incorporates into the DNA strand.
- ara-C 1- ⁇ -D-arabinofuranosylcytosine
- NAC counteracts the effects of oxidative stress by scavenging ROIs and increasing intracellular glutathione (GSH).
- GSH intracellular glutathione
- Previous studies have demonstrated that NAC is a potent inhibitor of phorbol ester-induced activation of the HIV-1 long terminal repeat. This antioxidant has also been found to inhibit activation of the nuclear factor ⁇ B (NF- ⁇ B) by phorbol esters and other agents such as H 2 O 2 .
- NF- ⁇ B nuclear factor ⁇ B
- ROIs are also formed during the treatment of cells with ionizing radiation. Thus, the cellular response to this agent may involve the ROI-induced activation of transcription factors and thereby longer term effects on gene expression.
- the HL-525 variant expresses relatively low levels of PKC ⁇ and PKC ⁇ compared to HL-205 cells. Low to undetectable levels of PKC ⁇ mRNA were also found in both the HL-205 and HL-525 lines (data not shown).
- PKC isozymes which are sensitive to PKC down-regulation may be responsible for transducing signals which confer x-ray inducibility of the c-Jun gene.
- prolonged TPA treatment could cause down-regulation of other PKC-independent signaling pathways involved in induction of c-Jun by ionizing radiation.
- X-ray treatment was previously shown to be associated with activation of a PKC-like activity.
- Clone HL-205 was isolated from the human HL-60 myeloid leukemia cell line.
- the phorbol ester-resistant variant of HL-60 cells designated HL-525, was isolated by exposing wild-type cells to low concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA; 0.5 to 3 nM) for 102 passages.
- TPA 12-0-tetradecanoylphorbol-13-acetate
- TPA 12-0-tetradecanoylphorbol-13-acetate
- FBS fetal bovine serum
- Irradiation was performed at room temperature using a Gamma cell 1000 (Atomic Energy at Canada Ltd., Ontario) with a 137 Cs source emitting at a fixed dose rate of 14.3 Gray (Gy)/min as determined by dosimetry.
- RNA was purified by the guanidine isothiocyanate-cesium chloride technique. The RNA was analyzed by electrophoresis through 1% agarose-formaldehyde gels, transferred to nitrocellulose filters, and hybridized to the following 32P-labeled DNA probes: 1) the 1.8-kb BamHI/EcoRI insert of a human c-Jun gene purified from a pBluescript SK(+) plasmid; 2) the 1.5-kb EcoRI fragment of the murine Jun-B cDNA from the p465.20 plasmid; 3) Jun-D; 4) the 0.9-kb ScaI/NcoI insert of human c-fos gene purified from the pc-fos-1 plasmid; 5) the 2.0-kb PstI insert of a chicken ⁇ -actin gene purified from the pA1 plasmid; and 6) the 1.9-kb B
- Hybridizations were performed at 42° C. for 24 h in 50% (v/v) formamide, 2 ⁇ SSC, 1 ⁇ Denhardt's solution, 0.1% SDS, and 200 ⁇ g/ml salmon sperm DNA.
- the filters were washed twice in 2 ⁇ SSC-0.1% SDS at room temperature and then in 0.1 ⁇ SSC-0.1% SDS at 60° C. for 1 hour.
- the digested DNAs were run in 1% agarose gels and transferred to nitrocellulose filters.
- Hybridizations were performed with 10 7 cpm of 32 P-labeled RNA/ml in 10 mM Tris-HCl, ph 7.5, 4 ⁇ SSC, 1 mM EDTA, 0.1% SDS, 2 ⁇ Denhardt's solution, 40% formamide, and 100 ⁇ g/ml yeast tRNA for 72 h at 42° C.
- the filters were washed in: a) 2 ⁇ SSC-0.1% SDS at 37° C. for 30 min; b) 200 ng/ml RNase A in 2 ⁇ SSC at room temperature for 5 min; and c) 0.1 ⁇ SSC-0.1% SDS at 42° C. for 30 min.
- Egr-1 enhancer-promoter was cloned into the XhoI and SacI restriction endonuclease sites of the luciferase reporter vector (Promega). This construct is illustrated in FIG. 1. Activation of the luciferase gene produces luminescence in extracts of transfected cells.
- DNA constructs were transfected into cells using Lipofectin (GIBCO BRL).
- DNA Egr-1-LUC
- GIBCO BRL Lipofectin solution
- Solution A For each plate to be transfected dilute 5-10 ⁇ l DNA into a final volume of 100 ⁇ l Opti-MEM 1 Medium.
- Solution B For each plate to be transfected dilute 5-50 ⁇ l of Lipofectin Reagent into a final volume of 100 ⁇ l Opti-MEM 1 Medium.
- tissue culture plate in 4 ml of the appropriate growth medium supplemented with serum.
- DNA (Egr-1-LUC) is mixed at a concentration of 1 ⁇ g/10 ⁇ l in Lipofectin solution (GIBCO BRL). Prepare a tube with 3 ml of cell culture medium.
- the treated cells were harvested from four wells for each time point.
- Radioactive 131 I was chosen because it is high energy beta emitting isotope, and also yields accessibility of radio-labeling to many proteins such as tumor specific monoclonal antibodies.
- results indicate that this isotope stimulates Erg-1 activity with results comparable to X-ray irradiation (4-10 Gy) in all three human pancreatic cancer cell lines.
- Results of the human Pancreatic cancer cell line AsPC-1 are shown in FIG. 2.
- Utilizing 6 MBq of 131 I exposure for 60 min. results in 11,706 RLU (relative light unit), at 3 hrs 35,648 RLU, and at 6 hrs the highest values of 81,508 RLU, while at 12 hrs there was a decline to 47,490 RLU. All PBS and NaOH controls ranged from 4224 to 9678 RLU.
- Tc99m radioisotope Technetium
- FIG. 3 shows the effects of higher doses of Tc99m on the human Pancreatic cancer cell line AsPC-1.
- a dose of 14.3 MBq is lower than the amount applied for clinical imaging.
- Adding 14.3 MBq of Tc99m to culture plate was associated with an RLU of 8,408 at 24 while backgrounds were 500 to 600 RLU for this experiment. Again, these results indicate that Tc99m can activate the Egr-1 promoter to express the LUC gene nearly ten to fifteen fold above background.
- Tc99m (14.3 MBq) on the all three human Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2 and Panc-1) transfected with the are listed in Table 1.
- the results indicate that Tc99m stimulates the Egr-I-LUC gene at 24 hours in all three cell lines.
- the increase in gene expression for AsPC-1 was 14-fold, for MIA PaCa-2 18 times and for Panc-1 was 26 times above background.
- a DNA molecule comprising a radiation responsive enhancer-promoter operatively linked to an encoding region that encodes a polypeptide or any other useful component, such as an antisense molecule with a sequence that is the antisense version to an oncogene.
- a construct that comprises a CArG domain of an Egr-1 promoter and the gene for tumor necrosis factor.
- the construct is then administered to the patient to be treated.
- This administration may be in the form of intravenous infusion of naked DNA, or through the use of viral delivery vectors.
- viral vectors are retrovirus that is self-inactivating, adenovirus, or adeno-associated virus. If a retrovirus is employed, lymphokine-activated killer (LAK) cells are first infected with the retrovirus containing the construct, then administered to the patient.
- LAK lymphokine-activated killer
- the radionuclide such as Technetium-99m Sodium Methylene Diphosphonate
- the preferred intravenous dosage is up to 4 mCi.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Abstract
Description
TABLE 1 ______________________________________ Polypeptide Source of Encoding DNA ______________________________________ TNF CETUS ricin CETUS p53 Dr. Vogelstein, Johns Hopkins University MnSOD Genentech Pseudomonas Dr. Steve Lory, Univ. exotoxin of Washington ______________________________________
TABLE 2 ______________________________________ Plasmid Designation Enhancer-Promoter Encoding ______________________________________ Region pE425-TNF CArG domain of Egr-1 TNF pE425-CAT CArG domain of Egr-1 CAT pE425-p53 CArG domain of Egr-1 p53 pE425-raf 301-1 CArG domain of Egr-1 raf 301-1 pE425-MnSOD CArG domain of Egr-1 MnSOD pE425- CArG domain of Egr-1 Gal4/VP16 Gal4/VP16 c-Jun-CAT c-Jun promoter CAT AP-1CAT AP-1 CAT ______________________________________
TABLE 3 __________________________________________________________________________ Encoded Use Polypeptide Application to Disease __________________________________________________________________________ Kill tumor cells Toxins Solid & TNF Hematologic Growth Factors Malignancies (IL-1-6, PDGF,FGF) Protect normal Lymphokines GCSF Solid & tissues from CMCSF Hematologic radiation and other Erythropoietin Malignancies cytotoxins Aplastic Anemic during cancer therapy Inhibit Metastasis NM23 Cancer Metastasis Tumor Suppressor Rb p53 Prevention of Gene Products Malignancy Following Standard Radio therapy and Chemotherapy Radiosensitization TNF Solid & Chemosensitization Hematologic (enhance routine Malignancies treatment effects) Correct Defects in Factor 8 Clotting Clotting Factors Disorders Introduce Streptokinase Myocardial Anticlotting Urokinase Infarction Factors CNS Thrombosis, Pheripheral Thrombosis Correct Defects Normal Hemoglobin Sickle Cell Characterizing Anemia Hemoglobinopathy Use Encoded Application to Disease Polypeptide Correct Deficiencies Nerve Growth Factor Alzheimer's Leading to Disease Neurodegenerative Disease Provide Treatment Insulin Diabetes Component for Diabetes Disease of DNA ERCC-1, XRCC-1 Ataxia Repair Telangiectasia Abnormalities Xeroderma Pigmentosum __________________________________________________________________________
TABLE 4 ______________________________________ TNF-α level (units/ml) Cell Line Origin Control X-ray ______________________________________ STSAR-5 MFH 0.4 >2.0 STSAR-13 Liposarcoma 0.0 0.34 STSAR-33 Ewing sarcoma 0.17 >2.0 STSAR-43 Osteosarcoma 0.41 1.3 STSAR-48 Neurofibrosarcoma 0.28 0.43 ______________________________________ TNF-α levels were measured in medium from confluent cell cultures (control) and in irradiated confluent cells (xray). TNFα levels increased as measured by the ELISA technique. MFH, malignant fibrous histiocytoma.
TABLE 5 ______________________________________ Effects of Radioactive Isotope Tc99m in Human Pancreatic Cell Lines on Egr-1-LUC Gene Expression Time AsPC-1 MIA PaCa-2 PANC Control ______________________________________ 45 min 1,797 2,401 2,895 521 90 min 1,640 3,738 1,345 536 3 hr 1,636 1,690 2,859 603 6 hr 2,920 4,341 1,550 629 12 hr 1,699 4,866 2,079 505 24 hr 8,408 10,934 15,951 612 48 hr 3,990 3,188 4,618 543 ______________________________________
Claims (16)
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/241,863 US5571797A (en) | 1994-05-11 | 1994-05-11 | Method of inducing gene expression by ionizing radiation |
AU26891/95A AU696210B2 (en) | 1994-05-11 | 1995-05-11 | Methods of inducing gene expression by ionizing radiation |
DE69535407T DE69535407T2 (en) | 1994-05-11 | 1995-05-11 | METHOD FOR INDUCING GENE EXPRESSION BY MEANS OF IONIZING RADIATION |
EP95922079A EP0759083B1 (en) | 1994-05-11 | 1995-05-11 | Methods of inducing gene expression by ionizing radiation |
AT95922079T ATE355381T1 (en) | 1994-05-11 | 1995-05-11 | METHOD FOR INDUCING GENE EXPRESSION USING IONIZING RADIATION |
CA002192813A CA2192813C (en) | 1994-05-11 | 1995-05-11 | Methods of inducing gene expression by ionizing radiation |
PCT/US1995/005959 WO1995031559A2 (en) | 1994-05-11 | 1995-05-11 | Methods of inducing gene expression by ionizing radiation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/241,863 US5571797A (en) | 1994-05-11 | 1994-05-11 | Method of inducing gene expression by ionizing radiation |
Publications (1)
Publication Number | Publication Date |
---|---|
US5571797A true US5571797A (en) | 1996-11-05 |
Family
ID=22912470
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/241,863 Expired - Lifetime US5571797A (en) | 1994-05-11 | 1994-05-11 | Method of inducing gene expression by ionizing radiation |
Country Status (6)
Country | Link |
---|---|
US (1) | US5571797A (en) |
EP (1) | EP0759083B1 (en) |
AT (1) | ATE355381T1 (en) |
AU (1) | AU696210B2 (en) |
DE (1) | DE69535407T2 (en) |
WO (1) | WO1995031559A2 (en) |
Cited By (51)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998022142A1 (en) * | 1994-01-10 | 1998-05-28 | The Board Of Regents Of The University Of Nebraska | Antisense oligonucleotide compositions for selectively killing cancer cells |
US5770581A (en) * | 1990-12-20 | 1998-06-23 | Arch Development Corp. | Gene transcription and ionizing radiation: methods and compositions |
WO1998040105A1 (en) * | 1997-03-10 | 1998-09-17 | Research Development Foundation | Photodynamic therapy generated oxidative stress for temporal and selective expression of heterologous genes |
US5817636A (en) * | 1990-12-20 | 1998-10-06 | Arch Development Corp. | Control of gene expression by ionizing radiation |
WO1999025385A1 (en) * | 1997-11-17 | 1999-05-27 | Imarx Pharmaceutical Corp. | A method of increasing nucleic acid synthesis with ultrasound |
US5948681A (en) * | 1996-08-14 | 1999-09-07 | Children's Hospital Of Philadelphia | Non-viral vehicles for use in gene transfer |
WO1999054444A2 (en) * | 1998-04-22 | 1999-10-28 | Inex Pharmaceuticals Corporation | Combination therapy using nucleic acids and radio therapy |
US5997898A (en) | 1995-06-06 | 1999-12-07 | Imarx Pharmaceutical Corp. | Stabilized compositions of fluorinated amphiphiles for methods of therapeutic delivery |
US6025365A (en) * | 1997-03-25 | 2000-02-15 | Arch Development Corp. | Chelerythrine and radiation combined tumor therapy |
US6033646A (en) | 1989-12-22 | 2000-03-07 | Imarx Pharmaceutical Corp. | Method of preparing fluorinated gas microspheres |
US6071495A (en) | 1989-12-22 | 2000-06-06 | Imarx Pharmaceutical Corp. | Targeted gas and gaseous precursor-filled liposomes |
US6071494A (en) | 1996-09-11 | 2000-06-06 | Imarx Pharmaceutical Corp. | Methods for diagnostic imaging using a contrast agent and a renal vasodilator |
US6088613A (en) | 1989-12-22 | 2000-07-11 | Imarx Pharmaceutical Corp. | Method of magnetic resonance focused surgical and therapeutic ultrasound |
US6117414A (en) | 1991-04-05 | 2000-09-12 | Imarx Pharmaceutical Corp. | Method of computed tomography using fluorinated gas-filled lipid microspheres as contract agents |
US6123923A (en) | 1997-12-18 | 2000-09-26 | Imarx Pharmaceutical Corp. | Optoacoustic contrast agents and methods for their use |
US6139819A (en) | 1995-06-07 | 2000-10-31 | Imarx Pharmaceutical Corp. | Targeted contrast agents for diagnostic and therapeutic use |
US6143276A (en) | 1997-03-21 | 2000-11-07 | Imarx Pharmaceutical Corp. | Methods for delivering bioactive agents to regions of elevated temperatures |
US6231834B1 (en) | 1995-06-07 | 2001-05-15 | Imarx Pharmaceutical Corp. | Methods for ultrasound imaging involving the use of a contrast agent and multiple images and processing of same |
US6315981B1 (en) | 1989-12-22 | 2001-11-13 | Imarx Therapeutics, Inc. | Gas filled microspheres as magnetic resonance imaging contrast agents |
US6414139B1 (en) | 1996-09-03 | 2002-07-02 | Imarx Therapeutics, Inc. | Silicon amphiphilic compounds and the use thereof |
US6416740B1 (en) | 1997-05-13 | 2002-07-09 | Bristol-Myers Squibb Medical Imaging, Inc. | Acoustically active drug delivery systems |
US6444660B1 (en) | 1997-05-06 | 2002-09-03 | Imarx Therapeutics, Inc. | Lipid soluble steroid prodrugs |
US6443898B1 (en) | 1989-12-22 | 2002-09-03 | Imarx Pharmaceutical Corp. | Therapeutic delivery systems |
US6521211B1 (en) | 1995-06-07 | 2003-02-18 | Bristol-Myers Squibb Medical Imaging, Inc. | Methods of imaging and treatment with targeted compositions |
US6528039B2 (en) | 1991-04-05 | 2003-03-04 | Bristol-Myers Squibb Medical Imaging, Inc. | Low density microspheres and their use as contrast agents for computed tomography and in other applications |
US6537246B1 (en) | 1997-06-18 | 2003-03-25 | Imarx Therapeutics, Inc. | Oxygen delivery agents and uses for the same |
US6548047B1 (en) | 1997-09-15 | 2003-04-15 | Bristol-Myers Squibb Medical Imaging, Inc. | Thermal preactivation of gaseous precursor filled compositions |
US6551576B1 (en) | 1989-12-22 | 2003-04-22 | Bristol-Myers Squibb Medical Imaging, Inc. | Container with multi-phase composition for use in diagnostic and therapeutic applications |
US20030082685A1 (en) * | 2001-04-06 | 2003-05-01 | WEICHSELBAUM Ralph R. | Chemotherapeutic induction of egr-1 promoter activity |
US20030086903A1 (en) * | 2001-11-02 | 2003-05-08 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US6579522B1 (en) | 2000-06-27 | 2003-06-17 | Genvec, Inc. | Replication deficient adenoviral TNF vector |
WO2002049501A3 (en) * | 2000-12-18 | 2003-09-04 | Univ Texas | Local regional chemotherapy and radiotherapy using in situ hydrogel |
US6638767B2 (en) | 1996-05-01 | 2003-10-28 | Imarx Pharmaceutical Corporation | Methods for delivering compounds into a cell |
US6743779B1 (en) | 1994-11-29 | 2004-06-01 | Imarx Pharmaceutical Corp. | Methods for delivering compounds into a cell |
US20040242523A1 (en) * | 2003-03-06 | 2004-12-02 | Ana-Farber Cancer Institue And The Univiersity Of Chicago | Chemo-inducible cancer gene therapy |
US6841537B1 (en) | 1998-04-22 | 2005-01-11 | Protiva Biotherapeutics Inc. | Combination therapy using nucleic acids and conventional drugs |
US20050171396A1 (en) * | 2003-10-20 | 2005-08-04 | Cyberheart, Inc. | Method for non-invasive lung treatment |
US20060234272A1 (en) * | 2005-03-31 | 2006-10-19 | The Regents Of The University Of California | Using gene panels to predict tissue sensitivity to ionizing radiation |
US20060257369A1 (en) * | 2003-11-14 | 2006-11-16 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US20070036748A1 (en) * | 2001-04-06 | 2007-02-15 | Weichselbaum Ralph R | Combination therapies for cancer |
US20070212298A1 (en) * | 2004-08-25 | 2007-09-13 | Prefix Suffix | Use of the combination comprising temozolomide and tnf-alpha for treating glioblastoma |
US20080076122A1 (en) * | 2006-09-26 | 2008-03-27 | The Regents Of The University Of California | Characterizing exposure to ionizing radiation |
US20110050070A1 (en) * | 2009-09-01 | 2011-03-03 | Cree Led Lighting Solutions, Inc. | Lighting device with heat dissipation elements |
WO2011100651A1 (en) * | 2010-02-12 | 2011-08-18 | Ovokaitys Todd F | A laser enhanced amino acid blend and use of same to regenerate active myocardial tissue |
US8084056B2 (en) | 1998-01-14 | 2011-12-27 | Lantheus Medical Imaging, Inc. | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
US10040728B2 (en) | 2014-06-06 | 2018-08-07 | Todd Frank Ovokaitys | Methods and compositions for increasing the bioactivity of nutrients |
US10202598B2 (en) | 2014-05-30 | 2019-02-12 | Todd Frank Ovokaitys | Methods and systems for generation, use, and delivery of activated stem cells |
US10384985B2 (en) | 2014-06-06 | 2019-08-20 | B.K. Consultants, Inc. | Methods and compositions for increasing the yield of, and beneficial chemical composition of, certain plants |
WO2021195218A1 (en) | 2020-03-24 | 2021-09-30 | Generation Bio Co. | Non-viral dna vectors and uses thereof for expressing gaucher therapeutics |
WO2021195214A1 (en) | 2020-03-24 | 2021-09-30 | Generation Bio Co. | Non-viral dna vectors and uses thereof for expressing factor ix therapeutics |
WO2024040222A1 (en) | 2022-08-19 | 2024-02-22 | Generation Bio Co. | Cleavable closed-ended dna (cedna) and methods of use thereof |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU4994596A (en) * | 1995-03-01 | 1996-09-18 | Cornell Research Foundation Inc. | Interdependent adenoviral vectors and methods of using same |
US6080728A (en) * | 1996-07-16 | 2000-06-27 | Mixson; A. James | Carrier: DNA complexes containing DNA encoding anti-angiogenic peptides and their use in gene therapy |
DE69736692T2 (en) * | 1996-07-16 | 2007-06-14 | Archibald James Mixson | Cationic vehicle: DNA complexes and their use in gene therapy |
DE102008048036A1 (en) * | 2008-09-19 | 2010-03-25 | Meurer, Heinrich, Dr. | Method for detecting ionizing radiation |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5128126A (en) * | 1989-04-11 | 1992-07-07 | Boehringer Ingelheim International Gmbh | Use of pharmaceutical compositions containing at least one cytokine for the systemic treatment of preneoplastic lesions |
WO1994006916A1 (en) * | 1992-09-11 | 1994-03-31 | Arch Development Corporation | Gene transcription and ionizing radiation: methods and compositions |
-
1994
- 1994-05-11 US US08/241,863 patent/US5571797A/en not_active Expired - Lifetime
-
1995
- 1995-05-11 AT AT95922079T patent/ATE355381T1/en not_active IP Right Cessation
- 1995-05-11 WO PCT/US1995/005959 patent/WO1995031559A2/en active IP Right Grant
- 1995-05-11 AU AU26891/95A patent/AU696210B2/en not_active Ceased
- 1995-05-11 DE DE69535407T patent/DE69535407T2/en not_active Expired - Fee Related
- 1995-05-11 EP EP95922079A patent/EP0759083B1/en not_active Expired - Lifetime
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5128126A (en) * | 1989-04-11 | 1992-07-07 | Boehringer Ingelheim International Gmbh | Use of pharmaceutical compositions containing at least one cytokine for the systemic treatment of preneoplastic lesions |
WO1994006916A1 (en) * | 1992-09-11 | 1994-03-31 | Arch Development Corporation | Gene transcription and ionizing radiation: methods and compositions |
Non-Patent Citations (57)
Title |
---|
B. Christy, et al., "DNA Binding Site of the Growth Factor-Inducible Protein Zif268", Proc. Natl. Acad. Sci, USA, 86: 8737-8741, 1989. |
B. Christy, et al., DNA Binding Site of the Growth Factor Inducible Protein Zif268 , Proc. Natl. Acad. Sci, USA , 86: 8737 8741, 1989. * |
D. Boothman, et al., "Identification and Characterization of X-Ray-induced Proteins in Human Cells", Cancer Research, 49: 2871-2878, 1989. |
D. Boothman, et al., Identification and Characterization of X Ray induced Proteins in Human Cells , Cancer Research , 49: 2871 2878, 1989. * |
D. Hallahan, et al., "Protein Kinase C Mediates X-ray Inducibility of Nuclear Signal Transducers EGR1 and JUN", Proc. Natl. Acad. Sci. USA, 88: 2156-2160, 1991. |
D. Hallahan, et al., "Tumor Necrosis Factor Gene Expression is Mediated by Protein Kinase C Following Activation by Ionizing Radiation", Cancer Research, 51: 4565-4569, 1991. |
D. Hallahan, et al., Protein Kinase C Mediates X ray Inducibility of Nuclear Signal Transducers EGR1 and JUN , Proc. Natl. Acad. Sci. USA , 88: 2156 2160, 1991. * |
D. Hallahan, et al., Tumor Necrosis Factor Gene Expression is Mediated by Protein Kinase C Following Activation by Ionizing Radiation , Cancer Research , 51: 4565 4569, 1991. * |
Datta et al., "Ionizing radiation activates transcription of the EGR1 gene via CarG elements," Proc. Natl. Acad. Sci. USA, 89(21) :10149-10153, Nov., 1992. |
Datta et al., "Reactive oxygen intermediates target CC(A/T) 6 GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation," Proc. Natl. Acad. Sci. USA, 90(6) :2419-2422, Mar., 1993. |
Datta et al., Ionizing radiation activates transcription of the EGR1 gene via CarG elements, Proc. Natl. Acad. Sci. USA , 89(21) :10149 10153, Nov., 1992. * |
Datta et al., Reactive oxygen intermediates target CC(A/T) 6 GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation, Proc. Natl. Acad. Sci. USA , 90(6) :2419 2422, Mar., 1993. * |
Gubits et al., "Expression of Immediate Early Genes After Treatment of Human Astrocytoma cells with Radiation and Taxol,"Int. J. Radiation Oncology Biol. Phys., 27(3) :637-642, Oct., 1993. |
Gubits et al., Expression of Immediate Early Genes After Treatment of Human Astrocytoma cells with Radiation and Taxol, Int. J. Radiation Oncology Biol. Phys ., 27(3) :637 642, Oct., 1993. * |
Hallahan et al (1989) Proc Natl Acad Sci USA 86: 10104 10107. * |
Hallahan et al (1989) Proc Natl Acad Sci USA 86: 10104-10107. |
International Search Report dated Dec. 8, 1995. * |
K. Alexandropoulos, et al., "v-Fps-responsiveness in the Egr-1 Promoter is Mediated by Serum Response Elements", Nucleic Acids Research, 20(9): 2355-2359, 1992. |
K. Alexandropoulos, et al., v Fps responsiveness in the Egr 1 Promoter is Mediated by Serum Response Elements , Nucleic Acids Research , 20(9): 2355 2359, 1992. * |
L. Witte, et al., "Effects of Irradiation on the Release of Growth Factors from Cultured Bovine, Porcine, and Human Endothelial Cells", Cancer Research, 49: 5066-5072, 1989. |
L. Witte, et al., Effects of Irradiation on the Release of Growth Factors from Cultured Bovine, Porcine, and Human Endothelial Cells , Cancer Research , 49: 5066 5072, 1989. * |
Luna et al., "Photodynamic Therapy Mediated Induction of Early Response Genes," Cancer Research, 54(5) :1374-1380, Mar., 1994. |
Luna et al., Photodynamic Therapy Mediated Induction of Early Response Genes, Cancer Research , 54(5) :1374 1380, Mar., 1994. * |
M. Brach, et al., "Ionizing Radiation Induces Expression and Binding Activity of the Nuclear Factor κB", The American Society for Clinical Investigation, Inc., 88: 691-695, 1991. |
M. Brach, et al., Ionizing Radiation Induces Expression and Binding Activity of the Nuclear Factor B , The American Society for Clinical Investigation, Inc ., 88: 691 695, 1991. * |
M. Gessler, et al., "Homozygous Deletion in Wilms Tumours of a Zinc-Finger Gene Identified by Chromosome Jumping", Nature, 343: 774-778, 1990. |
M. Gessler, et al., Homozygous Deletion in Wilms Tumours of a Zinc Finger Gene Identified by Chromosome Jumping , Nature , 343: 774 778, 1990. * |
M. Lambert, et al., "X-ray-Induced Changes in Gene Expression in Normal and Oncogene-Transformed Rat Cell Lines", Journal of the National Cancer Institute, 80(18): 1492-1497, 1988. |
M. Lambert, et al., X ray Induced Changes in Gene Expression in Normal and Oncogene Transformed Rat Cell Lines , Journal of the National Cancer Institute , 80(18): 1492 1497, 1988. * |
M. Sherman, et al., "Ionizing Radiation Regulates Expression of the c-jun Protooncogene", Proc. Natl. Acad. Sci. USA, 87: 5663-5666, 1990. |
M. Sherman, et al., "Regulation of Tumor Necrosis Factor Gene Expression by Ionizing Radiation in Human Myeloid Leukemia Cells and Peripheral Blood Monocytes", The American Society for Clinical Investigation, Inc., 87: 1794-1797, 1991. |
M. Sherman, et al., Ionizing Radiation Regulates Expression of the c jun Protooncogene , Proc. Natl. Acad. Sci. USA , 87: 5663 5666, 1990. * |
M. Sherman, et al., Regulation of Tumor Necrosis Factor Gene Expression by Ionizing Radiation in Human Myeloid Leukemia Cells and Peripheral Blood Monocytes , The American Society for Clinical Investigation, Inc ., 87: 1794 1797, 1991. * |
Nedwin et al (1985) Nucleic Acids Research 13: 6361 6373. * |
Nedwin et al (1985) Nucleic Acids Research 13: 6361-6373. |
R. Attar, et al., "Expression Cloning of a Novel Zinc Finger Protein That Binds to the c-fos Serum Response Element", Molecular and Cellular Biology, 12 (5): 2432-2443, 1992. |
R. Attar, et al., Expression Cloning of a Novel Zinc Finger Protein That Binds to the c fos Serum Response Element , Molecular and Cellular Biology , 12 (5): 2432 2443, 1992. * |
R. Weichselbaum et al., "X-Ray Sensitivity of Fifty-three Human Diploid Fibroblast Cell Strains From Patients with Characterized Genetic Disorders", Cancer Research, 40: 920-925, 1980. |
R. Weichselbaum et al., X Ray Sensitivity of Fifty three Human Diploid Fibroblast Cell Strains From Patients with Characterized Genetic Disorders , Cancer Research , 40: 920 925, 1980. * |
R. Weichselbaum, et al., "In Vitro Radiobiological Parameters of Human Sarcoma Cell Lines", Int. J. Radiation Oncology Biol. Phys., 15: 937-942, 1988. |
R. Weichselbaum, et al., "Radiation-Resistant and Repair-proficient Human Tumor Cells May Be Associated with Radiotherapy Failure in Head-and Neck-Cancer Patients", Proc. Natl. Acad, Sci, USA, 83: 2684-2688, 1986. |
R. Weichselbaum, et al., In Vitro Radiobiological Parameters of Human Sarcoma Cell Lines , Int. J. Radiation Oncology Biol. Phys., 15: 937 942, 1988. * |
R. Weichselbaum, et al., Radiation Resistant and Repair proficient Human Tumor Cells May Be Associated with Radiotherapy Failure in Head and Neck Cancer Patients , Proc. Natl. Acad, Sci, USA , 83: 2684 2688, 1986. * |
Reuland et al (1991) Nucl Med Biol 18: 121 125. * |
Reuland et al (1991) Nucl Med Biol 18: 121-125. |
S. Dalton, et al., "Characterization of SAP-1, a Protein Recruited by Serum Response Factor to the c-fos Serum Response Element", Cell, 68: 597-612, 1992. |
S. Dalton, et al., Characterization of SAP 1, a Protein Recruited by Serum Response Factor to the c fos Serum Response Element , Cell , 68: 597 612, 1992. * |
S. Qureshi, et al., "v-Src Activates Both Protein Kinase C-Dependent and Independent Signaling Pathways in Murine Fibroblasts", Oncogene, 6: 995-999, 1991. |
S. Qureshi, et al., "v-Src Activates Mitogen-Responsive Transcription Factor Egr-1 via Serum Response Elements", The Journal of Biological Chemistry, 266(17): 10802-10806, 1991. |
S. Qureshi, et al., v Src Activates Both Protein Kinase C Dependent and Independent Signaling Pathways in Murine Fibroblasts , Oncogene , 6: 995 999, 1991. * |
S. Qureshi, et al., v Src Activates Mitogen Responsive Transcription Factor Egr 1 via Serum Response Elements , The Journal of Biological Chemistry , 266(17): 10802 10806, 1991. * |
V. Sukhatme, et al., "A Zinc Finger-Encoding Gene Coregulated with c-fos During GFrowth and Differentiation, and After Cellular Depolarization", Cell, 53: 37-43, 1988. |
V. Sukhatme, et al., A Zinc Finger Encoding Gene Coregulated with c fos During GFrowth and Differentiation, and After Cellular Depolarization , Cell , 53: 37 43, 1988. * |
Weichselbaum et al (1992) Int J Radiation Oncology Biol Phys 24: 565 567. * |
Weichselbaum et al (1992) Int J Radiation Oncology Biol Phys 24: 565-567. |
Weichselbaum et al., "Radiation Induction of Immediate early Genes: Effectors of the Radiation-Stress Response," Int. J. Radiation Oncology Biol. Phys., 30(1) :229-234, Aug. 1994. |
Weichselbaum et al., Radiation Induction of Immediate early Genes: Effectors of the Radiation Stress Response, Int. J. Radiation Oncology Biol. Phys., 30(1) :229 234, Aug. 1994. * |
Cited By (78)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6315981B1 (en) | 1989-12-22 | 2001-11-13 | Imarx Therapeutics, Inc. | Gas filled microspheres as magnetic resonance imaging contrast agents |
US6479034B1 (en) | 1989-12-22 | 2002-11-12 | Bristol-Myers Squibb Medical Imaging, Inc. | Method of preparing gas and gaseous precursor-filled microspheres |
US6461586B1 (en) | 1989-12-22 | 2002-10-08 | Imarx Therapeutics, Inc. | Method of magnetic resonance focused surgical and therapeutic ultrasound |
US6443898B1 (en) | 1989-12-22 | 2002-09-03 | Imarx Pharmaceutical Corp. | Therapeutic delivery systems |
US6551576B1 (en) | 1989-12-22 | 2003-04-22 | Bristol-Myers Squibb Medical Imaging, Inc. | Container with multi-phase composition for use in diagnostic and therapeutic applications |
US6088613A (en) | 1989-12-22 | 2000-07-11 | Imarx Pharmaceutical Corp. | Method of magnetic resonance focused surgical and therapeutic ultrasound |
US6071495A (en) | 1989-12-22 | 2000-06-06 | Imarx Pharmaceutical Corp. | Targeted gas and gaseous precursor-filled liposomes |
US6033646A (en) | 1989-12-22 | 2000-03-07 | Imarx Pharmaceutical Corp. | Method of preparing fluorinated gas microspheres |
US6156736A (en) * | 1990-12-20 | 2000-12-05 | Arch Development Corporation | Gene transcription and ionizing radiation: methods and compositions |
US7041653B2 (en) | 1990-12-20 | 2006-05-09 | The University Of Chicago | Gene transcription and ionizing radiation: methods and compositions |
US20010006954A1 (en) * | 1990-12-20 | 2001-07-05 | WEICHSELBAUM Ralph R. | Gene transcription and ionizing radiation: methods and compositions |
US5817636A (en) * | 1990-12-20 | 1998-10-06 | Arch Development Corp. | Control of gene expression by ionizing radiation |
US5770581A (en) * | 1990-12-20 | 1998-06-23 | Arch Development Corp. | Gene transcription and ionizing radiation: methods and compositions |
US6528039B2 (en) | 1991-04-05 | 2003-03-04 | Bristol-Myers Squibb Medical Imaging, Inc. | Low density microspheres and their use as contrast agents for computed tomography and in other applications |
US6117414A (en) | 1991-04-05 | 2000-09-12 | Imarx Pharmaceutical Corp. | Method of computed tomography using fluorinated gas-filled lipid microspheres as contract agents |
WO1998022142A1 (en) * | 1994-01-10 | 1998-05-28 | The Board Of Regents Of The University Of Nebraska | Antisense oligonucleotide compositions for selectively killing cancer cells |
US6576220B2 (en) | 1994-03-11 | 2003-06-10 | Imarx Therapeutics, Inc. | Non-invasive methods for surgery in the vasculature |
US6743779B1 (en) | 1994-11-29 | 2004-06-01 | Imarx Pharmaceutical Corp. | Methods for delivering compounds into a cell |
US5997898A (en) | 1995-06-06 | 1999-12-07 | Imarx Pharmaceutical Corp. | Stabilized compositions of fluorinated amphiphiles for methods of therapeutic delivery |
US6521211B1 (en) | 1995-06-07 | 2003-02-18 | Bristol-Myers Squibb Medical Imaging, Inc. | Methods of imaging and treatment with targeted compositions |
US6231834B1 (en) | 1995-06-07 | 2001-05-15 | Imarx Pharmaceutical Corp. | Methods for ultrasound imaging involving the use of a contrast agent and multiple images and processing of same |
US6139819A (en) | 1995-06-07 | 2000-10-31 | Imarx Pharmaceutical Corp. | Targeted contrast agents for diagnostic and therapeutic use |
US6638767B2 (en) | 1996-05-01 | 2003-10-28 | Imarx Pharmaceutical Corporation | Methods for delivering compounds into a cell |
US5948681A (en) * | 1996-08-14 | 1999-09-07 | Children's Hospital Of Philadelphia | Non-viral vehicles for use in gene transfer |
US6414139B1 (en) | 1996-09-03 | 2002-07-02 | Imarx Therapeutics, Inc. | Silicon amphiphilic compounds and the use thereof |
US6071494A (en) | 1996-09-11 | 2000-06-06 | Imarx Pharmaceutical Corp. | Methods for diagnostic imaging using a contrast agent and a renal vasodilator |
AU731502B2 (en) * | 1997-03-10 | 2001-03-29 | Research Development Foundation | Photodynamic therapy generated oxidative stress for temporal and selective expression of heterologous genes |
WO1998040105A1 (en) * | 1997-03-10 | 1998-09-17 | Research Development Foundation | Photodynamic therapy generated oxidative stress for temporal and selective expression of heterologous genes |
US6143276A (en) | 1997-03-21 | 2000-11-07 | Imarx Pharmaceutical Corp. | Methods for delivering bioactive agents to regions of elevated temperatures |
US6025365A (en) * | 1997-03-25 | 2000-02-15 | Arch Development Corp. | Chelerythrine and radiation combined tumor therapy |
US6444660B1 (en) | 1997-05-06 | 2002-09-03 | Imarx Therapeutics, Inc. | Lipid soluble steroid prodrugs |
US6416740B1 (en) | 1997-05-13 | 2002-07-09 | Bristol-Myers Squibb Medical Imaging, Inc. | Acoustically active drug delivery systems |
US6537246B1 (en) | 1997-06-18 | 2003-03-25 | Imarx Therapeutics, Inc. | Oxygen delivery agents and uses for the same |
US6548047B1 (en) | 1997-09-15 | 2003-04-15 | Bristol-Myers Squibb Medical Imaging, Inc. | Thermal preactivation of gaseous precursor filled compositions |
US6716412B2 (en) | 1997-09-15 | 2004-04-06 | Imarx Therapeutics, Inc. | Methods of ultrasound treatment using gas or gaseous precursor-filled compositions |
WO1999025385A1 (en) * | 1997-11-17 | 1999-05-27 | Imarx Pharmaceutical Corp. | A method of increasing nucleic acid synthesis with ultrasound |
US6123923A (en) | 1997-12-18 | 2000-09-26 | Imarx Pharmaceutical Corp. | Optoacoustic contrast agents and methods for their use |
US8685441B2 (en) | 1998-01-14 | 2014-04-01 | Lantheus Medical Imaging, Inc. | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
US8658205B2 (en) | 1998-01-14 | 2014-02-25 | Lantheus Medical Imaging, Inc. | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
US8084056B2 (en) | 1998-01-14 | 2011-12-27 | Lantheus Medical Imaging, Inc. | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
US8747892B2 (en) | 1998-01-14 | 2014-06-10 | Lantheus Medical Imaging, Inc. | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
US9545457B2 (en) | 1998-01-14 | 2017-01-17 | Lantheus Medical Imaging, Inc. | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
WO1999054444A3 (en) * | 1998-04-22 | 1999-12-09 | Inex Pharmaceuticals Corp | Combination therapy using nucleic acids and radio therapy |
WO1999054444A2 (en) * | 1998-04-22 | 1999-10-28 | Inex Pharmaceuticals Corporation | Combination therapy using nucleic acids and radio therapy |
US6841538B1 (en) | 1998-04-22 | 2005-01-11 | Inex Pharmaceuticals Corporation | Combination therapy using nucleic acids and radio therapy |
US6841537B1 (en) | 1998-04-22 | 2005-01-11 | Protiva Biotherapeutics Inc. | Combination therapy using nucleic acids and conventional drugs |
US6579522B1 (en) | 2000-06-27 | 2003-06-17 | Genvec, Inc. | Replication deficient adenoviral TNF vector |
US20030175245A1 (en) * | 2000-06-27 | 2003-09-18 | Genvec, Inc. | Replication deficient adenoviral TNF vector |
US20050227910A1 (en) * | 2000-12-18 | 2005-10-13 | Yang David J | Local regional chemotherapy and radiotherapy using in situ hydrogel |
US7008633B2 (en) | 2000-12-18 | 2006-03-07 | Board Of Regents, The University Of Texas System | Local regional chemotherapy and radiotherapy using in situ hydrogel |
WO2002049501A3 (en) * | 2000-12-18 | 2003-09-04 | Univ Texas | Local regional chemotherapy and radiotherapy using in situ hydrogel |
US8034791B2 (en) * | 2001-04-06 | 2011-10-11 | The University Of Chicago | Activation of Egr-1 promoter by DNA damaging chemotherapeutics |
US20030082685A1 (en) * | 2001-04-06 | 2003-05-01 | WEICHSELBAUM Ralph R. | Chemotherapeutic induction of egr-1 promoter activity |
US20070036748A1 (en) * | 2001-04-06 | 2007-02-15 | Weichselbaum Ralph R | Combination therapies for cancer |
US7214368B2 (en) | 2001-11-02 | 2007-05-08 | Genvec, Inc. | Therapeutic regimen for treating cancer comprising the administration of adenoviral vectors comprising a TNF-α transgene |
US20070166284A1 (en) * | 2001-11-02 | 2007-07-19 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US20100305199A1 (en) * | 2001-11-02 | 2010-12-02 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US20030086903A1 (en) * | 2001-11-02 | 2003-05-08 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US20030086904A1 (en) * | 2001-11-02 | 2003-05-08 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US20040242523A1 (en) * | 2003-03-06 | 2004-12-02 | Ana-Farber Cancer Institue And The Univiersity Of Chicago | Chemo-inducible cancer gene therapy |
US20050171396A1 (en) * | 2003-10-20 | 2005-08-04 | Cyberheart, Inc. | Method for non-invasive lung treatment |
US20060257369A1 (en) * | 2003-11-14 | 2006-11-16 | Genvec, Inc. | Therapeutic regimen for treating cancer |
US20070212298A1 (en) * | 2004-08-25 | 2007-09-13 | Prefix Suffix | Use of the combination comprising temozolomide and tnf-alpha for treating glioblastoma |
US20110091375A1 (en) * | 2004-08-25 | 2011-04-21 | The University Of Chicago | Use of the combination comprising temozolomide and tnf-alpha for treating glioblastoma |
US20060234272A1 (en) * | 2005-03-31 | 2006-10-19 | The Regents Of The University Of California | Using gene panels to predict tissue sensitivity to ionizing radiation |
US20080076122A1 (en) * | 2006-09-26 | 2008-03-27 | The Regents Of The University Of California | Characterizing exposure to ionizing radiation |
US20110050070A1 (en) * | 2009-09-01 | 2011-03-03 | Cree Led Lighting Solutions, Inc. | Lighting device with heat dissipation elements |
WO2011100651A1 (en) * | 2010-02-12 | 2011-08-18 | Ovokaitys Todd F | A laser enhanced amino acid blend and use of same to regenerate active myocardial tissue |
US8404733B2 (en) | 2010-02-12 | 2013-03-26 | Todd F. Ovokaitys | Laser enhanced amino acid blend and use of same to regenerate active myocardial tissue |
US10907144B2 (en) | 2014-05-30 | 2021-02-02 | Todd Frank Ovokaitys | Methods and systems for generation, use, and delivery of activated stem cells |
US11905510B2 (en) | 2014-05-30 | 2024-02-20 | Todd Frank Ovokaitys | Methods and systems for activating cells to treat aging |
US10202598B2 (en) | 2014-05-30 | 2019-02-12 | Todd Frank Ovokaitys | Methods and systems for generation, use, and delivery of activated stem cells |
US10384985B2 (en) | 2014-06-06 | 2019-08-20 | B.K. Consultants, Inc. | Methods and compositions for increasing the yield of, and beneficial chemical composition of, certain plants |
US10865157B2 (en) | 2014-06-06 | 2020-12-15 | B.K. Consultants, Inc. | Methods and compositions for increasing the yield of, and beneficial chemical composition of, certain plants |
US10040728B2 (en) | 2014-06-06 | 2018-08-07 | Todd Frank Ovokaitys | Methods and compositions for increasing the bioactivity of nutrients |
WO2021195218A1 (en) | 2020-03-24 | 2021-09-30 | Generation Bio Co. | Non-viral dna vectors and uses thereof for expressing gaucher therapeutics |
WO2021195214A1 (en) | 2020-03-24 | 2021-09-30 | Generation Bio Co. | Non-viral dna vectors and uses thereof for expressing factor ix therapeutics |
WO2024040222A1 (en) | 2022-08-19 | 2024-02-22 | Generation Bio Co. | Cleavable closed-ended dna (cedna) and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
AU696210B2 (en) | 1998-09-03 |
WO1995031559A2 (en) | 1995-11-23 |
EP0759083B1 (en) | 2007-02-28 |
EP0759083A1 (en) | 1997-02-26 |
AU2689195A (en) | 1995-12-05 |
DE69535407D1 (en) | 2007-04-12 |
ATE355381T1 (en) | 2006-03-15 |
WO1995031559A3 (en) | 1996-01-11 |
DE69535407T2 (en) | 2007-11-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5571797A (en) | Method of inducing gene expression by ionizing radiation | |
US5770581A (en) | Gene transcription and ionizing radiation: methods and compositions | |
US5817636A (en) | Control of gene expression by ionizing radiation | |
Jäättelä et al. | Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. | |
US20090136465A1 (en) | Therapeutic Gene-Switch Constructs and Bioreactors for the Expression of Biotherapeutic Molecules, and Uses Thereof | |
JP2014512808A (en) | Vectors that conditionally express proteins | |
HUE024479T2 (en) | Engineered dendritic cells and uses for the treatment of cancer | |
US20030157494A1 (en) | Smooth muscle cell promoter and uses thereof | |
Datta et al. | Expression of the jun-B gene during induction of monocytic differentiation | |
CA2192813C (en) | Methods of inducing gene expression by ionizing radiation | |
JP2003500422A (en) | Osteonectin-based toxic gene therapy to treat calcified tumors and tissues | |
JP2004500884A (en) | Methods and means for regulating gene expression | |
AU2113299A (en) | Self-regulated apoptosis of inflammatory cells by gene therapy | |
WO2001030799A1 (en) | Methods to enhance and confine gene expression in cancer therapy | |
EP1139750A1 (en) | Bone sialoprotein based toxic gene therapy for the treatment of calcified tumors and tissues | |
JP2002529068A (en) | Methods of treating tumors using FAS-induced apoptosis | |
AU3470899A (en) | Inhibition of binding of hox and homeodomain-containing proteins and uses thereof | |
Dumortier | Cytokine gene transfer by adenoviral vectors as a novel therapeutic option for hepatitis B virus infection |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ARCH DEVELOPMENT CORPORATION, ILLINOIS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:WEICHSELBAUM, RALPH R.;REEL/FRAME:007122/0881 Effective date: 19940722 Owner name: ARCH DEVELOPMENT CORPORATION, ILLINOIS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KUFE, DONALD W.;REEL/FRAME:007122/0879 Effective date: 19940715 |
|
AS | Assignment |
Owner name: DANA-FARBER CANCER INSTITUTE, MASSACHUSETTS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNEE'S ADDRESS. PREVIOUSLY RECORDED ON REEL 7122, FRAMES 879;ASSIGNOR:KUFE, DONALD W.;REEL/FRAME:007203/0795 Effective date: 19940715 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
CC | Certificate of correction | ||
AS | Assignment |
Owner name: ARCH DEVELOPMENT CORP., ILLINOIS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:OHNO, TSUNEYA;REEL/FRAME:009922/0667 Effective date: 19990427 |
|
FEPP | Fee payment procedure |
Free format text: PAT HLDR NO LONGER CLAIMS SMALL ENT STAT AS NONPROFIT ORG (ORIGINAL EVENT CODE: LSM3); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
FEPP | Fee payment procedure |
Free format text: PAT HOLDER CLAIMS SMALL ENTITY STATUS, ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: LTOS); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 8 |
|
FPAY | Fee payment |
Year of fee payment: 12 |
|
REMI | Maintenance fee reminder mailed |